Registration Dossier

Administrative data

Endpoint:
toxicity to reproduction: other studies
Type of information:
experimental study
Adequacy of study:
other information
Study period:
march 06 - september 08 1992
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Basic data given (although not conform with the current guideline for reproduction toxicology)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996

Materials and methods

Principles of method if other than guideline:
The method is not a classical method for the testing of reproduction toxicity of chemicals as required by the OECD guideline. After treatment of animals (male), the testis was removed after sacrifice and subjected to gross- and histopathological scrutiny.
GLP compliance:
yes
Type of method:
in vivo

Test material

Constituent 1
Reference substance name:
o-toluidine hydrochloride
IUPAC Name:
o-toluidine hydrochloride
Constituent 2
Chemical structure
Reference substance name:
o-toluidinium chloride
EC Number:
211-252-8
EC Name:
o-toluidinium chloride
Cas Number:
636-21-5
Molecular formula:
C7H9N.ClH
IUPAC Name:
2-methylanilinium chloride
Details on test material:
- Name of test material: o-toluidine hydrochloride
- Analytical purity: 100%
- Lot/batch No.: Lot 308828/1 891

Test animals

Species:
rat
Strain:
other: F344/N rats
Sex:
male
Details on test animals or test system and environmental conditions:
- Source: Charles River Breeding Laboratories, Raleigh, NC
- Age at study initiation: 45d
- Weight at study begin: 153g
- Housing: 5 animals per cage
- Diet: NIH-07 Open Formula Diet (Zeigler Bros., Inc., Gardners, PA); feed was available ad libitum
- Water was available ad libitum
- Acclimation period: 9d


ENVIRONMENTAL CONDITIONS
- Temperature (°C): ca. 22
- Humidity (%): 50% ± 15%
- Air changes (per hr): at least 10/h
- Photoperiod: 12h per day

Administration / exposure

Route of administration:
oral: feed
Type of inhalation exposure (if applicable):
whole body
Vehicle:
other: test substance was administered via feed
Details on exposure:
Storage temperature of food:
Approximately 9% o-toluidine hydrochloride was lost during feed preparation. 5 mg/g o-toluidine hydrochloride-feed mixture were stored in sealed containers in the dark at room temperature, -20 C or 5 C for 7, 14, or 28 days or in an animal feeder, open to air and light, for 1 or 4 days. All samples were analyzed by HPLC. Feed samples stored in sealed containers in the dark at -20 or 5 C were stable for 28 days. The feed sample stored at room temperature for 28 days contained 90% of the o-toluidine hydrochloride detected in the sample before storage.
Details on analytical verification of doses or concentrations:
Analysis was performed by HPLC.
Duration of treatment / exposure:
13-Week interim groups: treatment with 0ppm, 5000ppm o-toluidine hydrochloride for 13 weeks
Stop-exposure groups: treatment for 13 weeks with 13 weeks recovery (0 ppm, 5000 ppm for first 13 weeks then 0 ppm o-toluidine hydrochloride for final 13 weeks
26-Week continuous-exposure groups: treatment with 0 ppm or 5,000 ppm o-toluidine hydrochloride for 26 weeks
Frequency of treatment:
continuous
Duration of test:
13 weeks, 26 weeks (treatment stop after 13 weeks), 26 weeks of continuous treatment
Doses / concentrations
Remarks:
Doses / Concentrations:
0 ppm and 5,000 ppm (approx. 375 mg/kg bw)
Basis:

No. of animals per sex per dose:
13-week interim:
Normal gastrointestinal flora groups: 10 control rats; 20 rats (o-toluidine hydrochloride)

Stop-exposure:
Normal gastrointestinal flora groups: 20 rats (o-toluidine hydrochloride)
Altered gastrointestinal flora groups: 10 control rats; 20 rats (o-toluidine hydrochloride)

26-week study:
Normal gastrointestinal flora groups: 10 control rats; 20 rats (o-toluidine hydrochloride)
Details on study design:
Rats were observed twice daily. Feed consumption was measured weekly. Mortality was checked twice a day. Body weights and clinical signs were recorded weekly and at necropsy. The testis and epididymis of all control rats and 10 of 20 rats from each exposure group were weighed at the end of each exposure Organs and tissues were examined for gross lesions. Histopathologic evaluations were performed on all rats at the 13-week interim evaluation and at the end of the studies. The testes was examined in all groups.
Statistics:
The Fisher exact test, a procedure based on the overal proportion of lesion-bearing animals, was used to evaluate histopathologic lesion data.
Armitage, (1971) Statistical Methods in Medical Research. John Wiley and Sons, New York.; Gart et al. (1979) J. Natl. Cancer Inst. 62, 957-974.

Organ and body weight data, which have approximately normal distributions, were analyzed with the parametric multiple comparison procedures of Dunnett (1955) J. Am. Stat. Assoc. 50, 1096-1121.


Results and discussion

Observed effects

NOAEL was not calculated. However, histopathological examination of the testis of the rats was performed.

BODY WEIGHT AND WEIGHT GAIN (mean body weight of rats in all exposed groups were lower than those in control groups) 13-week interim: 139g versus 175g Stop-exposure: 187g versus 224g 26-week study: 163g versus 224g

GROSS PATHOLOGY TESTIS AND EPIDIDYMIS:
13-week interim:
no gross lesions, absolute testis and epididymis weights slightly less than those of the control group (see table below). Degeneration of seminiferous tubules was a unilateral testicular lesion present in 5% to 10% of rats

26-week stop-exposure groups:
no gross lesions, absolute testis and epididymis weights slightly less than those of the controls groups. Degeneration of seminiferous tubules was a unilateral testicular lesion present in 5% to 10% of rats. 2 of 20 rats had epididymal mesothelioma

26 -weeks-continuous-exposure groups:
no gross lesions, absolute testis and epididymis weights slightly less than those of the controls group. Degeneration of seminiferous tubules was a unilateral testicular lesion present in 5% to 10% of rats. 1 rat had epididymal mesothelial cell hyperplasia

Any other information on results incl. tables

--Relative weight of testis was significantly increased with 5.07 versus  4.61 g after 13 weeks and 4.8 versus 4.2 g after 26-weeks
--Degeneration of seminiferous tubules was a unilateral
testicular lesion present in 5% to 10% of rats from each
exposed group. At 26 weeks, 2 of 20 rats had epididymal
mesothelioma (stop-exposure group) and one rat had
epididymal mesothelial cell hyperplasia
(continuous-exposure group).

OTHER: for more information see also chapters 5.4 and 5.7

Applicant's summary and conclusion