UKEMS genotoxicity trial 1981

Mutation Research, 100 (1982) 381-387

381

Elsevier Biomedical Press

UKEMS G E N O T O X I C I T Y T R I A L 1981 IN VIVO ASSAY SYSTEMS

J.C. TOPHAM (Coordinator) Imperial Chemical Industries Ltd., Pharmaceuticals Division, Safety of Medicines Department, Mereside, Alderley Park, Macclesfield, Cheshire SKIO 4TG (Great Britain)

(Received 24 July 1981) (Accepted 12 August 1981)

SUMMARY 4CMB was tested in 7 mouse micronucleus assays (3 i.p., 2 oral, 1 s.c., 1 foetal), 2 mouse sperm-head morphology assays (1 i.p., 1 s.c.), 1 dominant lethal test in mouse (oral), 3 SLRL tests in Drosophila (2 feeding, 1 injection) and one somatic cell segregation assay in Drosophila (feeding). 4 H M B was tested in 4 mouse micronucleus assays (2 i.p., 1 oral, 1 s.c.), 2 mouse sperm morphology assays (1 i.p. and 1 s.c.), 1 SLRL test and 1 somatic cell segregation assay in Drosophila (both feeding). BC was tested in 4 mouse micronucleus assays (2 i.p., 2 oral, 1 s.c.), 2 spermmorphology assays (1 i.p., 1 s.c.), 2 SLRL tests and 1 somatic cell segregation assay in Drosophila (all feeding). All the mouse assays gave negative results with all 3 compounds. The response of 4CMB and BC was not reproducible when tested under similar conditions in the SLRL test in Drosophila. All 3 compounds gave a positive response in the somatic cell segregation assay in Drosophila.

In vivo studies form a crucial part of any comprehensive evaluation of genotoxic potential. Only in vivo studies permit the full range of permeability, biotransformation and tissue-repair mechanisms to have their appropriate impact on the genotoxic potential assessment. In vivo studies have an important role in identifying agents that express a genotoxic potential in lower organisms or in isolated m a m m a l i a n cell systems but which are unable to reach the critical targets in Abbreviations: i.p., intra-peritoneal; s.c., sub-cutaneous; SCE, sister-chromatid exchange; SLRL, sex-

linked recessive lethal. 0165-1218/82/0000-0000/$02.75 © Elsevier Biomedical Press

382 TABLE 1 M I C R O N U C L E U S TESTS IN MICE Code

Strain

Route

Sex

Number

Sampling time (h)

of doses JB ND MR BHA MMcG JT RC a

CD-I TO CD-I(ICR)BR NMRI CD- 1 (CBA × Balb/c)Fi Porton

i.p. i.p. i.p. oral oral s.c. i.p.

c~ q~ cr c~ 9 ~ cr o" Q

5 1/2 2 2 1/2 1/2 1

6 24/6 6/24 6 48/72/96 24/6 30/48

aFl fetal tissue examined after administration to F0 female as well as maternal marrow.

Abbreviations: JB, Life Sciences, Stock; ND, University College of Swansea; MR, Huntingdon Research Centre; BHA, Leo Laboratories; MMcG, Inveresk Research International; JT, ICI Pharmaceutical Division; RC, University of Sussex; D M C D , University of Edinburgh; MF, Chester Beatty Institute; GW, ICI Central Toxicology Laboratory.

whole animals (e.g. methyl orange, sodium azide). Conversely, whole animal systems may also identify genotoxic agents which do not have readily demonstrable mutagenic potential in other systems (e.g. hexamethylphosphoramide, benzene, diethyl stilboestrol (de Serres and Ashby, 1981). In contrast, the usefulness of these assays as indicators of mutagenic potential in whole mammals depends on how closely they approach the ideal of identifying heritable consequences of exposure of F0 mammals in the F1 generation. None of the 4 in vivo assays used in this trial fulfill all these criteria. The assay systems used in this trial were the micronucleus test in mice, the F0 sperm-head morphology assay in mice, the dominant lethal test in mice, the sexlinked recessive lethal test in Drosophila (SLRL)and the somatic cell segregation assay in Drosophila. 7 micronucleus tests were conducted, each with a different protocol with respect to the route of administration, number of doses, tissue sampling time and sex of animals (Table t). The F0 sperm-head morphology assay was conducted after either s.c. or i.p. administration. The dominant lethal test in mice was conducted only with orally administered 4CMB. The protocols for these assays are summarised in Table 2. 2 sex-linked recessive lethal tests in Drosophila were performed using feeding/topical administration to larva or adult flies and one by injection of adults. The somatic cell segregation assay in Drosophila was conducted with feeding/topical administration to larva and embryos (see Table 3). The multiplicity of protocols employed, particularly in the micronucleus test, reflects the rapid development of these assays but also illustrate the difficulties in-

383 TABLE 2 DOMINANT LETHAL TEST 1N MICE Strain

Route

Sex

Number of doses

Sample time

AP

Oral

cr

5

Weekly × 4

Doses 5, 10, 25 mg/kg/day (4CMB only). SPERM-HEAD MORPHOLOGY IN MICE Strain

Route

Number of doses

Sample time

(CBA x Balb/c)F1

s.c. i.p.

5 5

35 days 35 days

c u r r e d in selecting the o p t i m u m p r o t o c o l to m a x i m i s e the o p p o r t u n i t i e s o f detecting g e n o t o x i c activity o f a c o m p o u n d with u n d e f i n e d p r o p e r t i e s . A beneficial o u t c o m e o f the wide range o f p r o t o c o l s e m p l o y e d was t h a t the c o m p o u n d s have been e v a l u a t e d u n d e r a very wide r a n g e o f c o n d i t i o n s . 4 C M B was a s s a y e d in 14 test systems, 4 H M B in 8 systems a n d BC in 9 systems (see T a b l e 4). 4 C M B was the only c o m p o u n d a s s a y e d in the d o m i n a n t lethal test in mice a n d in the t r a n s p l a c e n t a l m i c r o n u c l e u s test. A l l 3 c o m p o u n d s were a s s a y e d at least once in the m i c r o n u c l e u s test by 3 routes (oral, i.p. a n d s.c.), Fl s p e r m - h e a d m o r p h o l o g y by 2 routes (i.p. a n d s.c.) the S L R L test a n d the s o m a t i c cell m i s r e g u l a t i o n assays in D r o s o p h i l a ( b o t h by feeding).

RESULTS M o u s e assays N o n e o f the m o u s e assays gave a positive result with any o f the c o m p o u n d s tested. TABLE 3 DROSOPHILA ASSAYS Code

Strain

SLRL route

Stage

Sample time

DMcD

Oregon K

Oregon K Substrain

Adult Larvae Adult Larval + embryos

J Immediate J Post-treatment 0, 3, 9 days

MF

Feeding Feeding Injection Topical/feeding

Larval + embryos

10-20 days

Up to 15 days

Eye sectoring (soma) MF

Oregon K

Topical/feeding

384 TABLE 4 4CMB tested in all 14 systems

7 micronucleus (3 i.p.) (2 oral) (1 s.c.) (1 foetal) 2 sperm morphology (1 i.p.) (1 s.c.) 1 dominant lethal in mice (oral) 3 SLRL in Drosophila (2 feeding) (1 injection) 1 eye-sectoring misregulation in Drosophila (feeding) 4HMB (8 test systems)

4 micronucleus (2 i.p.) (1 oral) (1 s.c.) 2 sperm morphology (1 i.p.) (1 s.c.) 1 SLRL in Drosophila (feeding) 1 eye-sectoring misregulation in Drosophila (feeding) B C (9 test systems)

5 micronucleus (2 i.p.) (2 oral) (1 s.c.) 2 sperm morphology (1 i.p.) (1 s.c.) 2 SLRL in Drosophila (feeding) 1 eye-sectoring misregulation in Drosophila (feeding)

Although in some tests occasional data points were judged positive or achieved statistically significant differences from the negative controls, for example 72 h after oral administration a positive response was seen with 4CMB and 4 H M B in one micronucleus test and after both s.c. and i.p. dosing in the F0 sperm-head assay statistically significant increases were seen after high doses of all 3 compounds. However, none of these responses were reproducible, showed evidence of a dose-response relationship or completely fulfilled all the criteria required for the classification of a positive response for any of the chemicals.

Drosophila assays (a) Sex-linked recessive lethal test. In one SLRL test a small but statistically significant increase was found, following administration of all 3 compounds by the topical/feeding route to late embryos and newly hatched larvae at dose levels of 2 mM but 4CMB (the only c o m p o u n d tested at lower dose levels) had no effect at 1 mM. All 3 compounds were judged to be poorly mutagenic having induced a frequency of scorable mutations of about 0.6% which is about 3 times greater than the normal control level for this system. In this study approx. 2000 chromosomes were tested in each assay. In two other SLRL assays in which 4CMB and BC were fed to larvae and adults (4CMB was also injected into adults). No evidence of a mutagenic effect was obtained. In these studies 4CMB was fed at 3.6 m M and BC at 4.0 mM and approx. 1300 chromosomes were tested in each assay. In view of the non-reproducibility of the data and the absence of a dose-response

385

TABLE 5 4CMB TOXICITY Code

IN MICE

Strain

Route

Sex

Number

Dose

of doses

(mg/kg)

Lethality

LDs0 (mg/kg)

JB

CD-I

i.p.

o"

5

150

1/5

-

MR

CD-I(ICR)BR

i.p.

cr

2

120

No deaths

-

ND

TO

i.p.

c:

2

50

1/4

-

JT

(CBA × Balb/c)F1

i.p.

o"

5

50

2/5

-

DMcG AW

CD-l AP

Oral Oral

cr cr

2 5

25

4/18

71 -

JT

(CBA × Balb/c)Fl

s.c.

o"

5

500

2/5

-

JB

CD-1

i.p.

9

5

30

3/5

-

MR

CD-I(ICR)BR

i.p.

9

2

120

No deaths

-

BHA

NMRI

Oral


2

25

2/5

-

RC

Porton

i.p.

a <9

l

250

a0/3

-

a p r e g n a n t females: f o e t a l d e a t h s at 50 m g / k g .

relationship, 4CMB and BC cannot be regarded as positive in the sex-linked recessive lethal test and thus, must be classified as negative. As the assay with 4HMB has not been repeated but only gave a similar response for 4CMB at the single dose level tested 4HMB also cannot confidently be classified as positive in this assay. (b) Eye-sectoring assay. This assay (conducted in only one laboratory) measures the consequences of events in somatic cells in the emerging adult males at 2 different end-points. At high dose levels (0.5-2.0 mM) all 3 compounds induced effects at one end-point but clear effects were only seen with 4HMB and BC at the other end-point.

Toxicity o f 4CMB, 4HMB and BC to mice With all 3 compounds repeated administration by any route enhanced the observed toxic or lethal effects seen after single dosing. 4CMB. The lethality data on 4CMB accumulated in the course of the mouse studies is summarised in Table 5. Comparisons between studies are difficult because of differences in route, number of doses, sex and period of observation. In general 4CMB appeared to be more toxic to females and to have approximately equal toxicity when given by the oral or i.p. routes, subcutaneous administration being appreciably less toxic. However, there was some evidence that the variation seen between similar studies were at least in part due to differences in the efficiency with which 4CMB had been formulated which may lead to marked differences in absorption. In view of this variation no conclusion could be drawn concerning inter-strain differences in sensitivity to the toxic effects of 4CMB. What is important however, is that in all the

386

TABLE 6 4HMB TOXICITY Code

IN M I C E

Strain

Route

Sex

Number

Dose

of doses

(mg/kg)

Lethality

LDs0 (mg/kg)

JB

CD-1

i.p.

cy

5

400

1/3

-

MR

CD-I(1CR)BR

i.p.

o"

2

200

No deaths

-

ND

TO

i.p.

c¢

1/2

400

No deaths

-

JT

(CBA x Balb/c)Fl

i.p.

c~

5

400

5/5

856

MMcG

CD-I

Oral

o,

2

-

-

JT

(CBA x Balb/c)Fl

s.c.

~

5

1000

No deaths

-

MR

CD-I(ICR)BR

Oral

9

2

200

No deaths

-

laboratories except one 4CMB was tested up to lethal doses as defined under the conditions in each laboratory. 4HMB. 4HMB was significantly less toxic than 4CMB and there was less variation of data between laboratories (Table 6). In males the toxicity decreased according to route in the order i.p., oral, s.c. There were no deaths in the single study conducted in females and again the data does not allow any conclusions regarding strain differences in sensitivity, nor was there any evidence of variation in absorption between laboratories. In many of the studies lethality was not achieved although the doses employed were considerably higher (e.g. by at least a factor of 2) than in the corresponding studies with 4CMB. BC. BC was less toxic by the oral route than by the s.c. or i.p. route to male mice (Table 7). The limited data with females does not indicate any evidence of a sex difference in toxicity. In the majority of the studies, lethality was achieved. There was relatively little interlaboratory variation in the toxicity data. The lethality of BC was intermediate between 4CMB and 4HMB.

DISCUSSION

All the in vivo assays in mice gave uniformly negative results with all 3 compounds. In Drosophila in one laboratory 4CMB and BC were unequivocally negative in the SLRL test, but in the other laboratory using a similar protocol marginally positive results were obtained for all 3 compounds. In this same laboratory positive results in a somatic cell segregation assay in Drosophila were also obtained with all 3 compounds. In the mouse assays care was taken to ensure that the assays were conducted at the highest tolerated dose for the various strains of mice. Reproducibility and establishment of a dose-response relationship were an integral part of the criteria required for the classification of a positive response. There was an appreciation that occasional 'positive' data points will be found in results and care has been taken not to overinterpret data of this type.

387

TABLE 7 BC T O X I C I T Y IN MICE Code

Strain

Route

Sex

Number of doses

Dose (mg/kg)

Lethality

LDs0 (mg/ kg)

MR NO

CD- 1(ICR)BR TO

i.p. i.p.

o" cy

2 2

550 600

3/5 3/4

-

JT

(CBA z Balb/c)F1

i.p.

cr

5

200

2/5

-

DMcG JT MR BHA

CD-I (CBA x Balb/c)Fj NMRI

Oral s.c. i.p. Oral

cr cr 9 9

2 5 2 2

500 550 400

1/5 5/5 No deaths

1095 -

The mouse data indicate that none of the compounds were able to exert detectable genotoxic effects in the target cells (bone marrow, foetal liver, male germ cells) when given by several routes of administration up to the highest tolerated doses. Conflicting results in the SLRL test in Drosophila were found in the 2 laboratories, consequently there is no firm evidence for the induction of heritable mutation in this organism with 4CMB or BC and thus the results in this organism support conclusions drawn from the mouse data. All 3 compounds gave a positive response in the somatic cell segregation assay in Drosophila. This assay has not been widely used and the relationship between a positive response and potential oncogenic activity in mammals or the induction of heritable mutations in mammals has not yet been clearly established.

CONCLUSIONS

(1) There was no evidence of genotoxic activity in any of the mouse assays for any of the 3 chemicals when tested at doses which gave lethality by several routes of administration. (2) There was no evidence that genotoxic lesions can be induced in mouse germ cells by any of the 3 compounds. (3) There was a conflict of evidence of genotoxic activity in the SLRL test in Drosophila for both 4CMB and BC (and probably also for 4HMB). (4) All 3 compounds gave a positive response in the somatic cell segregation assay in Drosophila.

REFERENCE

de Serres, F.J., and J. Ashby (1981) Short-term tests for carcinogens: Report of the International Collaborative Program, Elsevier/North-Holland, Amsterdam.