A chronic toxicity and oncogenicity study in rats and subchronic toxicity study in dogs on ingested vinylidene chloride

FUNDAMENTAL AND APPLIEDTOXICOLOGY 3:55-02 (io83)

A Chronic Toxicity and Oncogenicity Study in Rats and Subchronic Toxicity Study in Dogs on Ingested Vinylidene Chloride J.F. QUAS'F, C.G. HUMISTON, C.E. WADE, J. BALLARD, J.E. BEYER, R.W. SCHWETZ and J.M. NORRIS Toxicology Research Laboratory, Healih and Environmental 5cienccs, U.S.A., Dew Chemical U.S.A., Midla,ld, MI 48640

ABSTRACT

A Chronic Toxicity and Oncogenicity Study in Rats and ,, Subchronic Toxicity Study in Dogs on Ingested Vinylidene Chloride. Quasi, J.F., Humiston, C.G., Wade, C.E., Ballard, J., Beyer, .I.E., Schwetz, R.W. and Norris, J.M. (1983).

Fundam. Appl. Toxicol. 3:55-62. The chronic toxicit) and oncogenic potential o f ingested vinylidene chloride ( V D C ) was evaluated in a 2-year study on Sprague-Dawley rats and

the subchronic toxicity was evaluated in beagle dogs in a 97-day study. The vinylidene chloride was incorporated in the drinking water of the rats at nominal concentrations of 50, 100 or 200 ppm. The time weighted average mg/kg body weight/day dosages of vinylidene chloride administered to the male and female rats over the 2-year period at the various mean anal) zed concentrations were 7, 10 or 20 for the males and 9, 14 or 30 for the females. Dogs were administered vinylidene chloride in peanut oil incorporated in a gelatin capsule at concentrations which provided 6.25, ! 2.5 or 25 mg vinyliden¢ chloride/kg body weight/day. There ~ere no significant differences between the groups of rats or dogs ingesting vinylidene chloride and their corresponding control groups in the following parameters: appearance and demeanor, mortality, body weight, food consumption, hematology, urinalysis, clinical chemistry determinations, organ weights and organ to body weight ratios. There were no significant differences in water consumption of the groups of rats ingesting vinylidene chloride and the controls. The sole treatment-related observation in the rats, evident only upon microscopic examination, was in the liver. The observation was characterized by a minimal amount of hepatocellular swelling with midzonal fatty change which occurred in the females at all dose levels and in the males only at the 200 ppm level. No exposure-related neoplastic changes occurred in the rats in any of the test groups. No exposure-related gross or histopathological changes were present in the tissues taken from the dogs at the termination of the 97-day study. INTRODUCTION Vinylidene chloride (1.1-dichloroethylene, CH2=CCI:, VDC) is used in the manufacture of various barrier resins and films for food packaging. Reports on the acute toxicity of vinylidene chloride in laboratory animals have indicated moderate toxicity by inhalation and by garage (Carpenter eta/., 1949; Rylova, 1953; Siegel et al., 1971; Jenkins et al.. 1972; Jaeger et al., 1973). Exposure to 100 ppm VDC for 6 weeks resulted in some loss of weight in rabbits and monkeys, but not in rats, guinea pigs, or dogs (Prendergast et aL, 1967). Rats exposed for up to 4 weeks to 500 ppm VDC vapor exhibited nasal irritation, decreased weight gain, and liver cell degeneration; at 200 ppm, the only sign of toxicity was slight nasal irritation (Gage, 1970).

Previous studies by Viola and Caputo (1977) and Maltoni et al. (1977) have shown that 75 to 100 ppm VDC vapor is noncarcinogenic in the rat. Maltom L.t a/. (1977} repotted that tu morJgenicity of 25 ppm VDC in the kidneys of male mice was associated with significant injury to that organ and that male mice were far more susceptible to the tomc effect of VDC than were the female mice. McKenna otal. (1977) have shown that the metabolism of VDC by male mice to electrophilic species which brad to collu. lar macromolecules correlated with the reported tumorigemc response in male mice. Recent stuclies reported by Roitz et aL ( 1 980) suggest the primary contributing factor in the induchon of the kidney tumors in the male mouse is cytotoxJcity as indicated by histopathology and is associated with a significant increase in DNA svnthasis. The objecttves of the studies reported herein wore to investigate the toxicological progerties of VDC Jn dogs and rats and to determine its oncogenlc potential following hfetime administration to rats. The VDC was administered by causule to the dogs and in the drinkmg water of rats. Incorporation in the drinking water was selected because the concentration could be held relatively constant in the water over a 24-hour perood whereas it was rapidly lost when incorporated in feed. ME. THODS Material The VDC samples used in the studies were aliquots of production material, minimum purity.99.5%, obiained from The Dew Chemical Company, Since the VDC used in the preparetion of various copolymers is distilled to remove the inhibitor monomet hyl ether of hydroquinone (MEHQ), the samples used in the studies were distilled to bring the residual MEHQ into the range of 1 to 5 ppm. The stability of VDC in water and peanut oil was verified before the initiation of the studies. Drinking water containing the VDC was prepared daily and analyzed at periodic intervals during the study. The means :t: S.D. of these analyses were 68 + 21 ppm, 99 + 22 ppm, and 2 0 6 + 33 ppm for the respective nominal concentrations of 50. 1O0 and 200 ppm VDC. Gelatin capsules for administration of the VDC to the dogs were filled weekly using the required amount of a 50% solution of VDC in peanut oil per capsule to provide the designated dosage on a mg/kg body weight basis. The dosages of 6.25, 12.5 and 25.0 were chosen to approximate 6250, 12 500 and 25 000 times, respectively, the amount of VDC ingested by a 75 kg person on a 1500 gram daily diet, assuming the food was in packages containing the compound and migration was 50 ppb in the food.

Copyfl|ht 1$liJ, .Societyof Tol.lcele|y

Fundamental and Applied Toxicology

r3j I-2/X3

55

QUAST. HUMISTON, WADE. BALLARD, BEYER. SCHWETZ A N D NORRIS I00-

Legend O PPM

SO PPM tO0 PPM 200 PPM

#."

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MONTHS ON STUDY FIC I. C~mm~d.~tive mort.dity o( male r,ds, m.~intained on dnnkllllg water conl.linizlg vinyhdrj'~e chloride.

Experimental design Male and female Sprague-Dawley rats, Spartan substrain) 6 to 7 weeks old, were randomly placed (2/cage) into suspended were bottom cages for use in the 2-year chronic toxicity and oncogenicity study. Mate and female pt~rebred beagle dogs. ;~ 8 months of age, were ~oused (4/pen) for use in the 97.day subchrome study. Food and water for the rats ~ and dog s~ were made available to the animals ad Ebltum. Groups of 48 rats/sex were maintained for up to 2 years on drinking water containing nominal concentrations of 50, 1O0 or 200 ppm VDC. A group of 80 rats/sex used as controls were supplied drinking water without VDC. Groups of 4 dogs/sex were administered one capsule containing VDC m peanut oil to provide dosages of 6.25, 12.5 or 25 mg VDC/kg body w e i g h t / d a y on each day of the 97-day study. A group of 4 control dogs/sex were daily administered e capsule containing an amount of peanut oil equivalent to that given to the dogs at the 25 mg/kg body weight dose level. The rats were observed at least twice weekly for changes in appearance or demeanor, signs of toximty and ewdence of neoplasm formation. Biweekly food consumption and daily water consumption were determined during the first 3 months; thereafter the determinations were made at periodic intervals. Body weights were ta:(en weekly for the first 14 weeks and then monthly thereafter, Hematologic determinations and urinalysis were conducted on at least 5 rats/sex/control and test groups at 6. ! 2. 18 and 23 months of the study. The packed cell volume, arythrocyte count, hemoglobin concentration, total ~Spat|.~n gc'~ra~¢h Animals, llm,,lell:, Michil'a':.. ~Marsh411 Research Animals. Inc.. ?~.~rth Ro,¢. Nov. Yohk. ~Pu~ma I.,abt}ratory Chow. Ral.',ton-Pulma Co., SI. Lores, Missouri. ' P u d n a Laboratory, Chc~g. Ralston-Purina Co.. St. Louts. Mis,,omi. [otliflcd u,Jth |q; peanut oil.

56

and differential leucocyte counts were determined:" Urmaiys=s included determination of specific gravity, p H sugar, protein. ketones, occult blood and bdir ubin. ~ Clinical chemistry studies were conducted on 5 rats/sex/control and test groups at 6, 12, and 18 months and from 10 rats/sex/group or from all survivors if less than ":O animals remained at terminal necropsy. The parameters monitored included blood urea nitrogen, serum alkaline phosphatase activity, serum glutamic gyrivic transaminase activity and glucose concentraliort r All rats that were found moribund or dead during the course of the study were subjected to gross pathological examination. At terminal necropsy following overnight del3rivation of food, the brain, heart, liver, kidneys, and testes were weighed. The following tissues were taken from rats dying spontaneously, found moribund or surviving until terminal necropsy: esophagus, salivary glands, stomach, large intestine, pancreas, liver, kidney, urinary bladder, prostate, accessory ~ex glands, epididymls, testes, ovaries, oviducts, uterus, mammary tissue, brain (cerebrum, cerebellum, brain stem), pituitary gland, spinal cord. peripl~eral nerve, trachea, lungs, nasal turb~nates. sternum and sternal bone marrow, ~pleen, mediastinal lymphcid tissue, lymph nodes (mesenteric). heart, aorta, skeletal muscle, adrenal glands, thyroid and parathyroid glands, adipose tissue, skin, eye and any gross lesions suggestive of a significant pathological alteration or neoplasm formation. Mmroscopic examinations were generally made on complete sets of tissues prepared by routine histological procedures and stained wlth hematoxylin and eosin, which were taken at the time of necropsy from all control and 200 13pro

" l .~ Melt1. Amchc.m ()phc.d Co.. Ihdlah,. %*¢~ York: Ihhl.=n,h~.. Amr~ C'n.. }'Ikdrl. lndlJ114, I ¢chn,¢,~n |nqltlmcm,. Corp.. |,It%~t¢,u,n. NCt¢ %'Ct'k I00,

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MONTHS ON STUDY FIG. 2. Cummulative mortality of female rats maintained on drinkinr; water containing vinylldene chloride.

Fundam. AppL ToxlcoL {3)

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VDC INGESTION STUDIES IN RATS AND DOGS

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DAYS ON S1UDY FIG. 3 Mean body weighI~ of rats maintamud t..n drinl, mg water cont.sin0ng vinylidenu ¢hloridu. dose level animals. Selected target organs and grossly recognized neoplastic changes in the rats receiving 1O0 or 50 ppm VDC in th~ drinking w a t e r were also examined m~cr oscopically. The dogs w e r e observed daily for signs of toxicity. Food c o n s u m p t i o n / p e n was recorded twice each week and the average amount c o n s u m e d / d o g / d a y was calculated. Hematologic determinations including packed cell volume, erythrocyte count, hemoglobin concentration, total and differential leukocyte counts w e r e conducted ~on all dogs prior to tile start o! the study and on test days 33 and 84. Urinalysis involwng specdic gravity, sugar, protein, ketonos, occult blood, bil=rubin ~ and microscopic examination of sediment were conducted on all dogs on test day 83, and pr=or to the t e r m m a l necropsy. Clinical chemistry d e t e r m i n a t i o n s of blood urea nqrogen, serum gluramie pyruvic transaminase activity, alkaline nhosphatase activity and serum glutamic oxaloecetic transaminase activ=ty w e r e made, f o l l o w i n g overnight deprivation of food. prior to the start of the study and suhsequentlythereafter at the same time urinalyses were conducted. The non-protein freP, sulfhydry[ content of a port{on of liver and kidneys w a s determined using the m e t h o d of Sedlak and Lindsay (1968) for each dog at the t i m e of necropsy. At the terminal sacrifice, following over-

night deprivation of food. the weights of the brain, heart, hver. kidneys and testes w e r e recorded. Portions of the same tissues and organs (except for the nasal turbinate} taken from tile rats on the chromc toxicity and oncogonicity Study wore taken from the dogs. M=croscopic examinat0on was conducted on a complete set of tissues from control and 200 ppm dose level an;reals and on liver, kidney and lung t=ssue from the 100 and 50 ppm dose tovol ammals. Ophthalmological e x a m m a h o n s w o r e conducted by slit lamp ophtl~almoscopy of all a m m a l s p~or to the start of the study and at the t e r m i n a t i o n of the study At t e r m i n a l necropsy, botll eyes of all dogs were examined groSSly and microscopic e x a m i n a t i o n was conducted on the eyes from control and top dose level dogs, Statistical evaluation of body w e i g h t and organ w m g h t data, food consumption (rats only), w a t e r consumphor~ (rats only). and hematology and chnical chem=stry determinahons were made us=ng an analysis of variance and Dunnett's test (Steel and Torrie, 1960). Since the dogs w e r e housed together regression analys~s was used to detect trcnds in food consumption, fn the rats all mortality and neoplasm incidence data w e r e analyzed using the Fisher Exact ProbabiliTy Test (Smgel. 1956). The level of significance m all cases w a s p <- 0.05.

TABLE "l Incidence of H i s t o p a t h o l o g i c a i l y D i a g n o s e d N e o p l a s m s in Rats I n g e s t i n g V i n y l i d e n e C h l o r i d e in t h e D r i n k i n g W a t e r

Content r-=hon of VDC in Water (ppm) 0 50 100 200

Fundmmental and Applied Tox|colog)

Total Number of Neoplasms Male

iO0 ~3 43 53

(3) 1-2183

Female

187 132 ]ZO 124

"l'ot,d Number Rats in Group Male

80 48 4B 47

Female

80 45 45 4~

Average Number of Neoplasmsl It,lt in Group Male

I3 1.1 0.o 1.1

[emale

2.3 28 25 2b

5"/

OUAST, HUMISTON, WADE, BALLARD, BEYER, SCHWETZ AND NORRIS

RESULTS Rats were administered VDC in the drinking water at nominal concentrations of 50. 100 or 200 ppm during the 2-year period. Based on the mean analyzed concentrations in the drinking water, the rats were determined to have ingested 7, 10 and 20 mg/kg/day, respectively, for the males and 9, 14 and 30 m g / k g / d a y , respectively, for the females. Throughout the 2 year study, the appearance and demeanor of thu rats ingesting VDC in their drinking water were not different from that of the control animals. Except for occasional statistically significant differences in the cumulative mortality percentages, the mortality among the test animals was comparable to the controls (Figures 1 and 2). Mean body weights of the rats over the 2-year period were similar for all groups (Figure 3). Occasionally occurring statisticaffy significant differences (increases and decreases) in food ana water consumption data were neither dose-related nor time-related and were not considered to be related to the ingestion of VDC.

increased incidence of hepatic changes was observed in the male rats in the 100 ppm group. No exposure-related hepattc changes were recognized in the 50 ppm group of male rats. Minimal hepatocellular fatty change and hepatocellular swelling were detected in female rats at all dose levels. No significant hepatocellular necrosis considered exposure.related w.3s evident in either male or female rats at any of the dose levels. Evaluation of the neoplasm data revealed an increased incidence of mammary gland fibroadenomas/adenofibromas in the females ingesting drinking water containing 50 ppm VDC. Since the incidence was within the normal range of the historical control data from this laboratory and was not dose-related. it was not considered to be related to the ingestion of VDC. The histopathologic diagnosis and number of neoplasms observed, including the mammary gland fibroadenomas/adenofibromas, is presented in Appendix Table 1. No other neoplastic types were observed which were significantly increased or decreased in either female or male rats. The total incidence of histopathologically diagnosed neoplasms, benign and malignant, is summarized in Table 1. The total neoplasm incidence in the male and female rats in ~,he various test groups was not differm~t than the incidence in the control animals.

All values for the hematological determinations and urinalyses obtained on animals from control and test groups were within the normal range for this laboratory. No consistent or Oose-rolated differences were observed in the clinical chemistry parameters or sulfhydryl levels evaluated and the various time periods in any of the test groups.

Dogs administered dady dosages of VDC of 6.25, 12.5 or 25 m g / k g / d a y over a 97-day period showed no oxposurerelateJ changes in appearance and demeanor, body weights or food consumption.

Mean organ weights, fasted boay weights and organ to body weight ratios were not significantly different for any of the test groups w h e n compared to the values for the control group. Tr~e only pathological findings considered to be related to the ingestion of VDC, evident only upon microscopic examination, were those involving the liver, The hepatic changes, w h e n present, were usually characterized b,/a minimal amount of mid-zonal hepatocellular fatty change in both male and female rats. In the male rats, only those in the 200 ppm group showed a statistically significantly increased incidence of hepatocellular fatty change. This group of rats also showed an increased incidence of hepatocellular swelling. A trend towards an

The hematologic values for packed cell volume, red blood cell count, total white-blood cell count and hemoglobin concentration are presented in Table 2. The mean w h i t e blood cell counts of the dogs receiving 6.25 and 25 mg/kg/day were significantly lower than the control values on day 84 of the study. Since the values for these groups of dogs were not different from their pre-exposure blood cell counts and were w i t h i n the range of no.'mal values for this laboratory, the differences were not considered to be related to the ingestion of VDC.

TABLE 2 M e a n H e m a t o l o g i c V a l u e s ^ f o r M a l e a n d F e m a l e Beagle D o g s G i v e n V i n y l i d e n e C h l o r i d e w i t h P e a n u t O i l O r a l l y in C a p s u l e s f o r 97 D a y s Dose Level (mgJkgsday) Days on Study:

Packed Cell Volume (%}

Red Blood Cells (× 10'lmm~ -48

33

Hemoglobin (gil00 mL) 84

-48

33

White Blood Cells (× 10~/mm j)

-4B

33

84

84

-48

33

84

0

51..z:3

51~I

50~2

b.q3~0.28

7.02~0.30

0o3"~.0.33

17.6-t-1,1 17.7~0.5

17.2"+0.0

130t2.2

13..5"+] 5

]2.9~:t.2

0.25

50:t:2

47"~2 47±2

b.oq.f0.33

0.O710.58

6.01-t-0.52

16.8:1:0.7 16.3"+0.9

16.4~:0.8

10.4.?.1,9

11.3~L3

10.7:I:0.6 h

9.8-t-1.0

l Z.0-.t: 1,9

10,8-f'0.S

.tfA L L'S

12..5

522.-4

50.t3

4o~2

6.89q- 0.4q

0.74"~'0.28

b.80"t-0.31

17.7-~1.0

17,1~0.8

17.1~0.7

25

50-~Z

48+-2

473:Z

6.q3:L0.34

6.90±0.31

6.80"~0.33

J7.2~1.0

]b.q51.1

t0.7:1:0.8

10.8:1:1.7 11.9:t:1.2

10.5-*t.6 a

FI'.'31A LL'S

0

50:t: |

49~3

48..+-1 b.33t-0.12

0.72~0.42

b.62~'0,3 ]

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17.2:t:0.5

0.7+0.5

10.1"1.4

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6.2.5

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4893

47~4

b.q3:l:0.o I

b.85~0.52

6.60"+'0.64

17.1-2:1.4

I b.b'*'0.8

16.3~-1.7

I 0.5±2.0

I0.q~1.8

10.7±3.2

12.5

48.~4

47~1

48"64

6.00.L0.52

6.62±0.21

6.761"0.50

16.7~1.5

16.5.'t.0.5

17.0"6"1.4 11.24-3.7

11.8-J:I.0

11,1-J:3.6

2.5

.50"66

45±3

48~0

b.52"~0.53

6.|8~0.26

6.07~0.60

17.0±1.8

15.b#I.l

1b.8"]:1.8

II 7+7..1

]1..5"~-2.1

II.7±2.2

^Valuee ,~re means :t: stand,lrd devi,stion for four dogs/group. BSigni~icant|y different from control mean by Dunnett's test, p < 0.05.

58

Fundsm. A ppl. ToxicoL (3)

January~February, 1983

VDC INGESTION STUDIES IN RATS AND DOGS TABLE 3 M e a n C l i n i c a l C h e m i s t r y V a l u e s ^ ( o r M a l e a n d F e m a l e Beagle D o g s G i v e n V i n y l i d e n e C h l o r i d e w i t h P e a n u t O i l O r a l l y in C a p s u l e s f o r o7 D a y s Dote Level

(mglkglday) Days on Study:

Serum Glutamie Pyruvic Transamlnate

Blood Urea Nitrogen

(rag %) -7

85

Alkaline Phosphat,Jse

(mU/mL)

Serum Glutamtc Oxa|oacettc Traosaminase

(mUImL)

q7

--7

~5

97

1~±2 18~4

42:t:b 4ba:b

34..1:1 421 I )

-10"24 702:20 p

722:1,q 85Z2]

-7

(mUtmL)

85

97

-7

85

07

bO:L2l Ol3"24

821.3o 10t~:L'7

30.15 40./;10

3,1~2 4218

31 :t:3 0,~3:21=t

M A I.ES

0 0.25

21:t0 21:~4

17:1:4 20.1:10

12.5

18¢2

15¢3

lb:r.2

.¢,l :L0

32Z" I l

40±7

b0¢25

71"_!:2o

75125

301.o

,aO'r o

3o.t8

25

17:1:1

18.I:4

20"~ 2,

4o:r5

122~ tbZ

54:I:10

b2~lZ

7tLZI

7~£1a

34~5

4115

42!8

22.1=2 172.2 ~

I71.3

I,~:t 3 21"2:2

42-2.8 3o:glO

38.1:7 50:Lb ~

4o:I.'8 542. I0

ot:I:15 8o.I:23

o45.:17 7,q ~:20

o5114 77:L21

3.t/=5 34~to

40:I.7 3412

44:±8 37:1.o

101.1:14

3o'28

38-e4

3o~:5

Oi4-t 4,5

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3817

4017

FI='MA L L'S

0 025

101:5

12.5

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181.3

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^Valut,~ awl. means :,-r.standard deviattt)n for four dog~,/grt,up.

I'Siglfi~¢tcantly d~flPrvnl Jrom cn/zztol mean by Dunnc/t't lest, p < 0.05

The clillical chem.stry values are presented in Table 3. Statistically significant differences found in the values of serum glutamic pyrivic transaminase, serum glutamic oxaloacetic transaminase and blood urea nitrogen for dogs receiving 6,25 mg/kg/day were within the range of normal values observed in this laboratory. Except for one male dog receiving 25 mg / k g / d a y of VDC that had an elevated serum glutamic pyr uric tr ansaminase activity on day 85 of the study but not on day 97, no alterations in the various determinations were observed at the 12,5 or 25 mg/kg/day levels. No alterations were observed in the non-protein free sulfhydryl levels in liver or kidney of dogs at any dose level. No exposure,related changes were observed in any of the parameters examined on urinalysis, in organ weights or organ to body weight ratios or in gross and microscopic examinat;on of the tissues from either the male or female dogs at any dose level. The pathologic changes present were interpreted to be spontaneous in occurrence and were comparable in control and test dogs. A single male dog receiving 12.5 mg/kg/day of VDC had kidneys which grossly appeared increased in size; however, no significant histopathologic alterations were recognized in this animal. Evaluation of hematologic, clinical chemistry, and urinalysis values of this dog did not reveal abnormalities. In view of these negative findings and the lack of other effects .at tile ,,25 mg/kg/day level, the grossly observed large kidneys in this one dog were considered not to be related to ingestion of VDC. DISCUSS/ON At the dose levels used in this 2-year study, VDC was found not to be oncogenic in either the male or female Sprague-Dawley iat. The highest concentration of VOC administered to the rats in the drinking water, 200 ppm. was the maximum feasible concentration based on the solubility of VDC in water. The only statistically significant treatment-related alteration, evident only upon microscopic examination, occurred in the liver, the target organ in the rat. After one year on study only the rats receiving the 200 pprn concentration were observed to have a minimal increase in cytoplasmic vacuolation of the hepatoFundamental and Applied Toxico~oly

f3) 1-2/83

cytes (Rampy e t a/., 19771. At the termination of the 2-year study, female rats at all concentrations of VDC in the drinking water and male rats at the 200 ppm concentration were observed to have no more than a minimal amount of hepatocellu[ar fatty change and hepatocellutar swelling. These minimal microscopic liver changes did not result in altered organ weight or clinical chemistry changes. The results of previous exper=ments demonstrated thai the fate of VDC in the rat following oral adminislratlon was dose dependent and the primary detoxification ~athway was the conjugation of VDC with hepatic glutathione (McKenna et al., 1978: Jaeger et eL, 1974; Jaeger et aL, 1973}, The maximum tolerated dose of VDC. defined as o.~e which results in pathologic alterations was 200 ppm in this rat study. Tile 200 ppm concentration did not deplete the non-protein sulfhydryl levels in the liver or the kidneys or cause toxicity by wrtue of saturation of tt3e detoxification pathway (Rampy et al.. t 977). At the highest dose level of VDC administered to dogs. 25 mg/kg of body weight/day, there likewise was no depletion of the non-protein sulfhydryl levels in the liver or kidney~ anJ also no exposure-related pathologic alterations in mate or female dogs at any dose level. In summary, the results of the 2-year study in rats indicate that ingestion of VDC at levels sufficient to induce alterations in the target organ, while not exceeding the capacity of the primary 0etoxification pathway, did not increase the overall incidence of neoplasms in this or any other organ. The rosuRs of the 97-day toxicity study in dogs demonstrated that 25 mg/kg/day, a level which did not alter the non-protein sulfhydryl levels in the liver or kidneys, did not elicit any evidence of toxicity. REFERENCES Carpenter, C.P., Sm~tth, H.F., Jr. and Pozzant, U.C. (104q). The assay of acute vapor toxicity, and the grading and Interpretation of results on ninety-six chemical compounds. J. lad./I)g. ToxicoL 31:3~3-346.

Gage, J.C. (1970), T h e s u b a c u t e inhalation toxicity of ] 0 q i n d u s trial chemicals. Brit..I. Ind, Med. 27:1-18.

59

QUAST, HUMISTON, WADE, BALLARD, BEYER, SCHWETZ AND NORRIS Jaeger, R.J., Conolly, R.B. and Murphy, S.D, (1973). Diurnal variation of hepatic glutathione concentration and its correlation with I,l-dichloroethylene inhalation toxicity in rats. Res. Commun. Chem. PathoL PharmacoL 6(2):465-471. Jaeger, R.J., Conolly, R.B. and Murphy, S.D. (1974). Effect of 18-hour fast and glutathione depletion on 1,1-dichtoroethyleneinduced hepatotoxicity and lethality in rats. Exp. Molec. PathoL 20:187-198. Jenkins, L.J., Jr., Trabulus, M.J. and Murphy, S.D. (1972). Biochemical effects of 1,1-dichloroethylene in rats: comparison with carbon tetrachloride and 1,2-dichloroethylene. Toxicol. AppL Pharmacal. 23:501-510. Maltoni, C , Cotti, G., Morisl, L. and Chieco, P. (1977). Carcinogenicity bloassays of vlnylidene chloride:Research plan and early results. La Medh:i,a del Lavoro 68(4):241-262. McKenna, M.J., Watanabe, P.G. and G e h r i n g , P.J. (I 977). Pha rmacokinetics of vlnylidene chloride in the rat. Environ. Ileahh Perspcc. 21:99-105. McKenna, M.J., Zempel, J.A., Madrid, E.O., Braun, W.H. and G e h r i n g , P.J. (1978). Metabolism and pharmacokinetic profile of vinylidene chloride in rats following-oral administration. 7bxicoL Apld. PharmacoL 45:821-835. Prendergast, J.A., Jones, R.A., Jenkins, L.J., Jr. and Siegel, J. (1967}, Effects on experimental animals of long-term inhalation of trichloroethylene, carbon tetrachloride, l , l , l - t r i c h l o r o ethane, dichlorodifluoromethane, and 1,1-dichloroethylene. 7bxicoL Apld. PharmacoL 10:270-289.

Rampy, L.W., Quast, J.F., Humiston, C.G., Balmer, M.F. and Schwetz, B.A. (1977). Interim results of two-year toxicological studies in rats of vinylidene chloride incorpora ted in the drinking water or administered by repeated inhalation. Environ. Ilealth Per~Tmc. 21:33-43. Reitz, R.H., Watanabe, P.G., McKenna, M.I., Quast, J.F. and G e h r i n g , P.I. (1980). Effects of vinylidene chloride on DNA synthesis and D N A repair in the rat and mouse: A comparative study with dimethylnitrosamine. 7bxicoL AppL PharmacoL 52:357-370. Rylova, M.L. (1953). Toxicity of l , l - e t h y l e n e dichloride. FarmakoL i. Toksiko116.47-50. Sedlak, !. and Lindsay, R.M, (1968). Estimation of total, proteinbound and nonprotein sulfhydryl groups in tissue with EIIman's Reagent. Anal. Biochem. 25:192-205. Siegel, S. (1956). Non-Parametric Statistics .for the Behavioral Sciences. McGraw-Hill Book Company, Inc., New York, New York. Siegel, J., Jones, R.A., Coon, R.A. and Lyon, J.P. (1971). Effects on experimental animals of acute, repeated, and continuous inhalation exposures todichloroacetylene mixtures. ToxicoL AppL PharmacoL 18:168-174. Steel, R.G.D. and Torrie, H.H. (1960). Principles and Procedures o f Statistics. McGraw-Hill Book Company, Inc., New York, New York. Viola, P.L. and Caputo, A. (1977). Ca rcinogenicity studies on vinyli, dene chloride. Environ. Ifealth Perspec. 21:45-47.

A P P E N D I X TABLE 1 H i s t o p a t h o l o g i c D i a g n o s i s a n d N u m b e r of N e o p l a s m s i n Rats I n g e s t i n g V i n y l i d e n e C h l o r i d e i n t h e D r i n k i n g W a t e r a Sex:

50

100

200

0

50

100

200

Total Number of Rats:

80

48

48

47

80

48

48

48

80

48

48

47

80

48

.48

48

1 2

o 0

0 ]

0 1

1

1

0

2

0

0

0

I

80

,18

48

47

80

48

48

48

0

0

1

0

0

0

0

0

80

48

48

47

80

47

48

48

1 1 0 O

0 2 o 0

0 0 0 0

0 0 0

o

o

o

o

o I

1 o

0

o o 1

o

o

o 1 0

63

5

2

39

72

2

1

41

1 1 0

1 1 I

0 1 0

1 0 0

0 2

0 o

0 1

1 3

0

0

0

0

69

48

48

46

73

18

46

46

0

0

0

0

0

0

1

0

Hyperplasic loci (neoplastic nodules) Hepatocellular carcinoma LUNG - Number examined

Bronchio-alveolar adenoma KIDNEY- Number examined

Embryonal nephroma (nephroblastoma) without metastasis Adenoma Hemangioma Renal tubular adenocarcinoma with pulmofiary metastasis TONGUE- Number examined

Keratoacanthoma without metastasis Squamous cell carcinoma of tongue Without metastasis Squamous papilloma ESOPHAGUS- Number examined

cell carcinoma without metastasis

S A L I V A R Y GLAND - Number examined

Medullary carcinoma wffhout metastasis Hemangioma in salivary gland region STOMA CH- Number examined

Leiomyosarcoma without metastasis Leiomyosarcoma with hepatic and mesenteric lymph node metastasis

66

Females

0

LIVER - Number exalnined

Squamous

Males

Conc. of VDC in Water (ppm):

63

4

1

32

69

o

2

43

1 0

0 0

0 0

0 0

0 0

0 0

0 0

0 1

80

42

45

47

79

17

~5

48

1

0 1

C 0

0

0 0

0 0

0 0

0 0

0

0

Fundam. A ppL ToxieoL (3)

January/February, J983

VDC

INGESTION

AND D O G S

S T U D I E S IN R A T S

A P P E N D I X T A B L E 1 (Cont'd.) Histopathologic Diagnosis and Number of Neoplasms in Rats Ingesting Vinylldene Chloride in the D r i n k i n g Water ^ Sex:

SMALL

Females

Cone. of VDC in W a t e r (ppm);

o

S0

100

2o0

0

50

100

200

Total N u m b e r o f Rats:

80

48

48

47

80

48

48

48

77

11

13

45

79

2

4

46

2 o 1 0

0 0 0 1

1

0

0 0 0 0

0 0 0 0

1 0 0 0

1 0 l 0

0 0 0 0

N u m b e r examined

INTESTINE-

Males

Mucinous cystadenocarcinoma without metastasis Mucinous cystadenocarcinoma with metastasis to large i nt e s t i ne Leiomyosarcoma w i t h o u t metastasis Leiomyosarcoma with metastasis to liver

l 0

77

6

6

45

77

0

2

40

0

0

0

0

0

0

I

0

- N u m b e r examined

78

45

29

47

7b

47

48

47

Pancreatic acinar adenoma (exocrine) Pancreatic islet cell adenoma

9 I0

7 2

3 3

6 8

0

0 l

0 2

I 7

79

21

19

46

2

I

0

0

75

19

19

45

1

0

0

0

79

30

28

44

0 I 0

2 0 0

0 0 0

0 0 1

79

29

27

46

21 3 0 0 0 o l

14 0 0 0 I 2 0

0 0 0 0 0 0 0

12 0 1 | 0 o 0

I

0

I

0

LARGE

INTESTINE-

N u m b e r examined

Carcinoma w i t h o u t m e t a s t a s i s PANCREAS

TI='STI:'S- N u m b e r examined

Interstitial cell adenoma - unilateral E P I D I D Y M I S - N u m b e r examined

Focal m e s o t h e l i o m a OVARY- N u m b e r examined Granulosa cell t u m o r w i t h o u t m e t a s t a s i s Leiomyoma Lipoma of m e s o v a r i u m U T E R U S - N u m b e r examined

Endometrial polyp formation Endometrial adenocarcinoma w i t h o u t metasta s i s Fibroma of the cervix Squamo us papilloma of e n d o m e t r i u m Stroma[ sarcoma Leiomyosarcoma w i t h o u t metastasis Endometrial medullary carcinoma with metast a s i s to uri na ry bladder, lymph nodes and small intestine Undifferentiated sarcoma, probably neurofibrosarcoma of u t e r u s and cervix M A L I G A ' A N T S C I I IVA N N O M A S (VARIOUS PRIMARY SITES) B R A I N - N u m b e r examined

Astrocytoma Olig odendrioglioma S P I A ' A L C"~ORD - N u m b e r examined

Glioblastoma, m a l i g n a n t w i t h o u t metastasis PERIPIIERA

L NERVES

- N u m b e r examined

Undifferentiated sarcoma - probable neurofib ros a rc oma without metastasis P I T U I T A R Y - N u m b e r examined

Adenoma Carcinoma with or w i t h o u t invasion of brain ADRENAL

GLAND-

N u m b e r examined

Adenoma of adrenal cortex Ad[enal gland carcinoma Adrenal pheochromocy!oma, unilateral Adrenal p h e o c h r o m o c y t o m a , bilateral Malignant a d r e n a l p h e o c h r o m o c y t o m a w i t h no a p p a r e n t me t a s t a s i s

Fundamental and Applied T o x i c o l o g y

f3) 1-2/83

5

.n

3

0

1

o

l

2

o

I

70

47

24

46

79

4~

47

48

1 0

0 !

0 0

0 0

0 0

0 0

0 0

I 0

74

0

]

45

75

0

0

,Io

0

0

o

I

0

0

0

0

73

0

0

41

69

0

0

45

Z

0

o

o

0

0

0

0

62

36

21

30

70

44

40

42

10 I

6 0

6 0

7 1

25 4

22 2

18 2

14 !

80

47

27

44

80

47

48

48

0 l

0 0

0 0

0 0

1 0

1 0

0

0

18 8 2

11 1 1

5 6 0

11 3 0

5

1

0 1

0 1

I

2

I

o

o

o

0

o

6!

QUAST, HUMISTON,

W A D E , BALLARD, BEYER, S C H W E T Z

AND

NORRIS

A P P E N D I X TABLE 1 ( C o n i ' d . ) H i s t o p a t h o l o g i c D i a g n o s i s a n d N u m b e r o f N e o p l a s m s i n R a t s I n g e s t i n g V i n y l t d e n e C h l o r i d e t n t h e .D r i n k i n g W a t e r ^ Males

Sex:

Females

Conc. of VDC In Water (ppm):

0

50

100

2.00

o

50

lO0

200

T o t a l N u m b e r o f Rats:

80

48

48

47

B0

48

48

48

76

45

29

45

75

47

47

48

5

4

2

1

0

0

1 0

0 0

1 0

5 0 o 0

5 1 o I

7 0 o o

6 I o o

S 1 0 0

414 0

414 0

212 0

0

111

0

0

0

0

0

0

0

413 0 111 0 0

0 1/1 0 0 111

0 0 0 0 0

0 0 0 111 0

0 0 0 0 0

212

111

414

0

0

0 0 0

111 0 0

0 0

0 0

0 0 0 o

94153 0 414 o

o

o

0

0

0

o 111

211

0

0

0

0

0

111

0

0

111

111

II1

;/l

Ill

111

1/1

0

0

0

0

0

0

1t1 0

0 0

0 0

0 0

111 0

0 0

0 1/1

0 0

18

10

7

14

20

10

14

10

1

1

0

0

0

0

0

0

S P L E E N - N u m b e r examined

80

45

26

45

79

4[5

47

48

Splenic hemangioma

]

0

0

0

0

0

0

0

75

46

45

43

77

46

46

46

I

0

1

0

0

0

0

0

75

46

45

43

77

46

46

46

1

l 0 0 1

0

2

0

0

0

0 0 0

3 0 0 0

o 0 0

o 0 0

o I 0

I 0 0

0 0 0

46

16

12

27

68

5

7

38

0

0

0

]

0

0

0

0

0 0 0 0

0 0 0 0

0

0

0

1

0

I

0

0

0

0

1 0

0 0

0 0

0

0 0 0

0 0

0

0 0 0 1 1

TIt YR OID - N u m b e r examined

C-cell adenoma (unilateral or bilateral) C-cell carcinoma without metastasis C-cell carcinoma with metastasis to lungs Follicular adenoma S U B C U T A N E O U S TISSU~ c

Fibroma Lipoma Malignant fibrous histiocytoma without metastasis Malignant fibrous histocytoma with lymph node involvement Fibrosarcoma with metastasis to mediastinum 3 ,tA MMA R Y G L A N D c

Fibroadenomaladenofibroma Adenoma Carcinoma without metastasis Papillary duct adenoma Fibrosarcoma Carcinosarcoma without metastasis

67140 n 64136 0 111 111 2t2 o II1

64135 0 0 0

E A R CA N:IL c

Sebaceous s q u a m o u s cell carcinoma of external auditory canal without metastasis (Zymbal gland tumor) Sebaceous s q u a m o u s cell carcinoma of external auditory canal "with metastasis to lungs M U S C U L O S K E L E T A L S YS TEMc

Osteogenic sarcoma without metastasis Undifferentiated fibrosarcoma of rear leg without metastasis E Y E - N u m b e r examined

Retrobulbar fibrosarcoma without metastasis

T I I Y M U S - N u m b e r examined

T h y m o m a with or without local invasion t . ; ' M P I t N O D F S - N u m b e r examined

Generalized lymphosarcoma w i t h widespread metastasis Lymphosarcoma of hind limb region without metastasis Lymphosarcoma of mesenteric lymph nodes Hemangioma of mesenteric lymph node BLOOD I : E S S E L S - N u m b e r examined

Aortic body t u m o r - chemodectoma MISCELLA NEOUS

HARD PALATE - Squamous celt carcinoma without metastasis HARD PALATE - Keratoacanthoma without metastasis ABDOMINAL WALL - Myxofibr0sarcoma without metastasis INTRAABDOMINAL fibrosarcoma PREPUTIAL GLAND - Squamous cell carcinoma without metastasis

^Except as noted, not significantly d i f f e r e n t f r o m control data w h e n analyzed usine Fischer'~ E~act Prnhahilitv Tp~t. n <' n n.~

SHot applicable. CData listed as n u m b e r at n e o p l a s m s / n u m b e r of rats w i t h neoplasm. All gross masses were examined microscopically. nSignificantly different from control data when analyzed usin~ Fischer's Exact Probability test, v < 0.05. 62

Fundam. April Toxicoi. (3)

Januark./Februar¥, 1983