Isobutyl 4-chloro-3,5-diaminobenzoate

Carcinogenicity

Administrative data

Endpoint:

carcinogenicity: dermal

Remarks:

subcutan

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Until June 1974

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

study sufficiently documented to support information from genetic toxicity studies

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1974

Report date:

1974

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: In an orienting carcinogenesis study, rats were dosed with the test item.
- Short description of test conditions: Rats were injected doses of the test item of 500 - 1000 mg/kg in intervals of 1 - 5 weeks subcutaneously.
- Parameters analysed / observed: Body weight gains, behaviour, appearance, survival time, localisation and amount of tumours.

GLP compliance:

no

Remarks:

conducted prior to GLP implementation

Test material

Test material information

Constituent 1

Reference substance name:

Isobutyl 4-chloro-3,5-diaminobenzoate

EC Number:

251-311-5

EC Name:

Isobutyl 4-chloro-3,5-diaminobenzoate

Cas Number:

32961-44-7

Molecular formula:

C11H15ClN2O2

IUPAC Name:

2-methylpropyl 3,5-diamino-4-chlorobenzoate

Test material form:

solid

Specific details on test material used for the study:

OTHER SPECIFICS: LD50 of the aqueous suspension with single subcutaneous application was >5000 mg/kg in female Wistar-W.64 rats weighing 120-140 g (Breeder Winkelmann, Borchen)

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

SPF-bred Wistar-W.64 rats

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Breeder Winkelmann, Borchen
- Age at study initiation: 100 days
- Housing: conventionally, in groups of 3 in Macrolon cages
- Diet (e.g. ad libitum): Altromin-Standardfutter ad libitum
- Water (e.g. ad libitum): tap water ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23±2°C

Administration / exposure

Route of administration:

subcutaneous

Details on exposure:

The test item was milled and, as suspension in physiological saline, injected subcutaneously in the middle of the back. Dependent of the state of the animals, the test item was injected in doses of 500 or 1000 mg/kg bw in intervals of 1 - 5 weeks. After 371 days the test item was applied a last time, a total dose of 25 g/kg was reached.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

371 days

Frequency of treatment:

intervals of 1 - 5 weeks

Post exposure period:

until death

Doses / concentrations

Dose / conc.:

25 other: g/kg

Remarks:

total dose, single doses of 500 or 1000 mg/kg

No. of animals per sex per dose:

18 (test item)
25 (control)

Control animals:

yes, concurrent vehicle

Positive control:

not required

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes

BODY WEIGHT: Yes

OTHER: survival time

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not specified

Mortality:

mortality observed, non-treatment-related

Description (incidence):

see below

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

see below

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

see below

Details on results:

Details can be derived from attached figures.
The average survival time of the male rats treated with isobutyl 4-chloro-3,5-diaminobenzoate was 824 ± 194 days of the study (As testing started at an age of 100 days, 100 days need to be added to the days of study to calculate total lifetime of the rats, which applies also to the following information on lifetime). Of the 18 male rats, 6 died with each a malignant tumour and 2 rats with each 3 benign tumours.
The average survival time of the female rats treated with isobutyl 4-chloro-3,5-diaminobenzoate was 737 ± 236 days of the study. Of these 18 female rats, 4 died with in total 5 malignant tumours. Further, in 8 animals in total 14 benign tumours were observed.
So of the 36 isobutyl 4-chloro-3,5-diaminobenzoate-treated rats 10 died with malignant tumours (28%). Total amount of malignant tumours were 11 (referring to the total animal number 31%), the benign 17 (47%).
The average survival time of the male control rats treated with physiological saline was 834 ± 229 days of the study, the one of the females 888 ± 177 days. Of the 25 male control rats 4 died with each one malignant tumour. Of the 25 female control rats 9 died with malignant tumours, 3 of them with a double tumour. Further, in 9 animals 14 benign tumours were observed. Of the 50 control rats hence 13 (26%) died with malignant tumours. The total number of benign tumours were 15 (30%).
For the used rat strain it is characteristic that the animals (especially females) develop when aging a high percentage of tumours of the hypophysis of questionable malignancy. In this case such tumours were formed in 19% of the treated animals and 40% in the control animals.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

>= 25 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

gross pathology

histopathology: neoplastic

mortality

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

compared to control

Applicant's summary and conclusion

Conclusions:

The available study was conducted scientifically reasonably. The study and the report do not provide equivalent information to serve as a standalone carcinogenicity study, but the given information suffices to support information from the available genotoxicity studies and to clearly indicate that isobutyl 4-chloro-3,5-diaminobenzoate does not induce tumours over background in Wistar-W.64 rats. So, isobutyl 4-chloro-3,5-diaminobenzoate does not need to be regarded as carcinogen.

Executive summary:

In an orienting carcinogenesis study, 100 d old Wistar-W.64 rats were injected doses of isobutyl 4-chloro-3,5-diaminobenzoate of 500 or 1000 mg/kg as suspension in physiological saline in intervals of 1 - 5 weeks subcutaneously in the middle of the back. After 371 days the test item was applied a last time, a total dose of 25 g/kg was reached, and the animals were observed until death. Examined parameters were Body weight gains, behaviour, appearance, survival time, localisation and amount of tumours. All dead animals were carefully dissected and tumours / suspected tumours histologically examined.

The average survival time of the male rats treated with isobutyl 4-chloro-3,5-diaminobenzoate was 824 ± 194 days of the study. Of the 18 male rats, 6 died with each a malignant tumour and 2 rats with each 3 benign tumours. The average survival time of the female rats treated with isobutyl 4-chloro-3,5-diaminobenzoate was 737 ± 236 days of the study. Of these 18 female rats, 4 died with in total 5 malignant tumours. Further, in 8 animals in total 14 benign tumours were observed. So of the 36 isobutyl 4-chloro-3,5-diaminobenzoate-treated rats 10 died with malignant tumours (28%). Total amount of malignant tumours were 11 (referring to the total animal number 31%), the benign 17 (47%)

The average survival time of the male control rats treated with physiological saline was 834 ± 229 days of the study, the one of the females 888 ± 177 days. Of the 25 male control rats 4 died with each one malignant tumour. Of the 25 female control rats 9 died with malignant tumours, 3 of them with a double tumour. Further, in 9 animals 14 benign tumours were observed. Of the 50 control rats hence 13 (26%) died with malignant tumours. The total number of benign tumours were 15 (30%).

Despite a total dose of 25 g/kg there were neither an elevated number of malignant tumours with a specific localisation nor tumours at the injections site which is more susceptible towards carcinogenic noxa. Weight gains, behaviour, appearance and survival times do also not indicate any impairment by the substance.

So, isobutyl 4-chloro-3,5-diaminobenzoate does not need to be regarded as carcinogen.