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Key value for chemical safety assessment

Effects on fertility

Description of key information

No effects on fertility and developmental toxicity were observed in a reproduction/developmental toxicity screening study in rats according to OECD guideline 421 (NOAEL 1000 mg/kg bw/day, the highest dose tested). A testing proposal has been made for a developmental toxicity study (OECD 414).

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 29 Oct 2003 to 01 May 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
27 July 1995
GLP compliance:
yes
Limit test:
no
Justification for study design:
according to guideline
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Raleigh, NC, USA
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) 9 wks
- Weight at study initiation: (P) 201-300 g
- Fasting period before study: no
- Housing: individually before mating, 1 male/1 female during mating
- Diet: Purina Certified Rodent Chow (No. 5002, PMI Feeds, Inc., St. Louis, MO, USA, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):22 +/-3
- Humidity (%): 30-70
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: As dose formulations at 2 and 200 mg/ml were stable for 35 days of storage in amber bottles in the refrigerator or ambient temperature, doses were formulated twice during the study (October 29, 2003, and November 25, 2003), dosing volume 5 ml/kg bw

VEHICLE
- Concentration in vehicle: 10, 50 and 200 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg bw
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged individually

Any female that did not show evidence of successful mating after 14 days of cohabitation was weighed weekly, and treatment continued until gd 26 or delivery occurred. If a female without a confirmed gd 0 date was, in fact, pregnant and delivered a litter, her lactational information was collected as described below.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis performed on day of preparation
Duration of treatment / exposure:
Males: 10 days prior to mating, throughout a 2 weeks mating period (necropsy thereafter)
Females: further treatment throughout gestation until lactation day 4
Frequency of treatment:
daily
Details on study schedule:
- Age at mating of the mated animals in the study: approx. 12 weeks (9 weeks + 1 week acclimation + 2 weeks of treatment)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations checked: observations included, but were not limited to, changes in skin and fur, eyes, mucous membranes, respiratory and circulatory system, autonomic and central nervous system, somatomotor activity, and behavior pattern.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: weekly, dams additionally on lactational days 0 and 4

FOOD CONSUMPTION:
recorded weekly with exception of the cohabitation period
Oestrous cyclicity (parental animals):
no
Sperm parameters (parental animals):
Parameters examined in male parental generation: testis weight, epididymis weight
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, fetal weight, physical or behavioural abnormalities, anogenital distance (AGD)

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals after the mating period
- Maternal animals: All surviving animals at day 4 postnatal

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]
Epididymides (pair), Testes (pair), Prostate, Seminal vesicies with coagulating glands and their fluids (pair), Ovaries (pair), Uterus with cervix and vagina,

HISTOPATHOLOGY / ORGAN WEIGHTS
Organs as above weighed and examined microscopically
Postmortem examinations (offspring):
GROSS NECROPSY
- Gross necropsy of dead pups
Statistics:
Treatment groups were compared to the concurrent control group using either parametric ANOVA (analysis of variance) under the standard assumptions or robust regression methods. The homogeneity of variance assumption was examined via Levene's Test. If it indicated lack of homogeneity of variance, robust regression methods were used to test all treatment effects. They were used to test for overall treatment group differences, followed by individual tests for exposed vs. control group comparisons (via Wald Chi-Square Tests), if the overall treatment effect was significant. If Levene's Test did not reject the hypothesis of homogeneous variances, standard ANOVA techniques were applied. The GLM procedure in SAS® 8.0 was used to evaluate the overall effect of treatment and, when a significant treatment effect was present, to compare each exposed group to the control via Dunnett's Test. For the litter-derived percentage, the ANOVA was weighted according to litter size. A one-tailed test (Dunnett's Test) was used for all pairwise comparisons to the vehicle control group, except that a two-tailed test was used for parental and pup body weight and organ weight parameters, feed consumption, and percent males per litter. Frequency data, such as reproductive indices, were not transformed. All indices were analyzed by Chi-Square Test for Independence for differences among treatment groups. When Chi-Square revealed significant differences, then a Fisher's Exact Probability Test was used for pairwise comparisons between each treatment group and the control group. Acquisition of anogenital distance was also analyzed by analysis of covariance (ANCOVA; in addition to ANOVA analysis). For correlated data, SUDAAN® software (Shah et al., 1997) was used for analysis of overall significance, presence of trend, and pairwise comparisons to the control group values.
Reproductive indices:
Females: Mating Index (%): No. females mated/No. females paired x 100
Fertility Index (%): No. females pregnant/No. females mated x 100
Gestational Index (%): No. females with live litters/No. females pregnant x 100

Males: Mating Index (%): No. males mated/No. males paired x 100
Fertillty Index (%): No. males siring litters/No. males mated x 100
Pregnancy Index (%): No. pregnant females/ No. males mated x 100

Offspring viability indices:
Live birth index (%): No. live pups at birth/Total No. pups born x 100
4-Day survival index (%): No. pups surviving 4 days/Total No. live pups at birth x 100
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Males: One male was recorded as ataxic at 1000 mg/kg bw/day on the first 2 days of dosing. Since this finding was transient and occurred only in one animal, the finding was considered not to represent true ataxia and was considered a response to the dosing procedure. A high incidence of postdose rooting exhibited a dose-response pattem of incidence. "Postdose rooting" and salivation are considered to be behavioral responses to taste aversion to the dosing formulations and not a toxic sign.
Females: Ataxia was recorded for 5 females at 1000 mg/kg bw/day alter dosing on the first two days. As noted above for the males, this observation was transient and considered not to be true ataxia. Therefore, these findings were considered to be a response to the dosing and not true ataxia. A high incidence of postdose rooting exhibited a dose-response pattern of incidence. "Postdose rooting" and salivation are considered behavioral responses to taste aversion to the dosing formulations and not a toxic sign.
Mortality:
mortality observed, treatment-related
Description (incidence):
One unscheduled death occurred in the FO females at 1000 mg/kg bw/day. The unscheduled death in the FO female was considered treatment¬related due to dystocia in late pregnancy.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males: There were significant reductions in FO male body weight at 1000 mg/kg bw/day for study days (sd) 7, 14, 21, and 27. At 1000 mg/kg bw/day, the body weight gainwas reduced for sd 0 to 7 and for the entire dosing period.
Females: There were no significant differences among groups for FO female body weights during the prebreed or mating (sd 0-28) periods. At scheduled sacrifice, mean body weights of FO females were equivalent across all dose groups. Body weight gain was significantly reduced at 1000 mg/kg/day for the period of sd 0 to 7 and from gd 0-7.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males: There were reductions in FO male feed consumption (expressed as g/day and g/kg/day) for sd 0 to 7 at 1000 mg/kg bw/day, and an increase from sd 7 to 14 at 1000 mg/kg bw/day.
Females: FO female feed consumption (expressed as g/day or g/kg/day) was significantly reduced at 1000 mg/kg bw/day for sd 0 to 7.
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):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Males: Histopathological findings included inflammation of the prostate gland in both the control and high-dose males. These changes were typical of the spontaneous type of microscopic pathology that can be observed at this age and in this strain of rat, and were not considered treatment related.
Females: No effects
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
There were no singnificant differences in FO mating, fertility, or pregnancy indices during the production of Fl offspring. Mating, fertility, and pregnancy indices were statistically equivalent across groups, and gestational length and precoital interval were equivalent across all groups. There were no significant differences across groups for percent preimplantation or postimplantation loss per litter or the number of dead pups at birth (pnd 0)
Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
System:
other:
Organ:
other: effects on body weight and feed consumption
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Clinical signs:
no effects observed
Description (incidence and severity):
There were no treatment-related clinical findings at any dose level.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Live birth and stillbirth indices were unaffected, as were the survival indices for pnd 0-4. The mean number of live pups per litter for pnd 0 and 4 was unaffected across all groups.F1 pup clinical observations during lactation indicated that the Fl pup mortality for pnd 0-4 was 2, 4, 6, and 4 pups at 0, 50, 250, and 1000 mg/kg bw/day, respectively.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean F1 pup body weights per litter (sexes combined or separately) were unaffected by treatment on pnd 0 and 4 across all dose groups
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
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Necropsy findings of F1 pups found dead on pnd 0-4 included pups that died on pnd 0 exhibiting open (fetal state) or closed (postnatal state) ductus arteriosus, no air (fetal state) or air (postnatal state) in lungs, no or little milk in the stomach, and autolysis. One pup that died on pnd 4 at 250 mg/kg/day was normal, and 1 at 1000 mg/kg bw/day was too autolyzed to evaluate.
Histopathological findings:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
The sex ratio (percent male pups/litter) was unaffected at any dose for any interval.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: absence of treatment-related adverse effects
Key result
Critical effects observed:
no
System:
other: no effects observed
Organ:
other: no effects observed
Key result
Reproductive effects observed:
no

Analytical verification of dose formulations showed that they contained 92.3 -102 % of the nominal concentration. Dose formulations at 2 and 200 mg/ml were stable for 35 days of storage in amber bottles in the refrigerator or ambient temperature.

Conclusions:
Under the conditions of this study, the parental LOAEL and NOAEL were 1000 and 250 mg/kg bw/day, respectively, based on reduced body weights and food consumption. No effects were oberved with respect to reproductive toxicity and in offspring up to 1000 mg/kg bw/day, the highest dose tested.
Executive summary:

Male and female CD® [Sprague-Dawley (SD)] rats (the F0 generation) were administered 1-chloro-2,3-dimethylbenzene orally by gavage at 0, 50, 250, and 1000 mg/kg bw/day at a dose volume of 5 ml/kg bw/day in Mazola® corn oil, 10 animals/sex/dose, for 2 weeks of prebreed exposure. After the 2-week prebreed exposure period, animals were randomly mated within treatment groups for a 2-week mating period to reproduce. Both sexes were exposed continuously through breeding and the females through 3 weeks of gestation and 4 days of lactation.

One female at 1000 mg/kg bw/day experiencing dystocia was euthanized moribund on gestational day (gd) 22; no other deaths occurred during conduct of the study. No other clinical signs were considered to be related to treatment. Body weights and weight change for the F0 males were decreased at 1000 mg/kg bw/day throughout the study although weight gain was similar to controls after the first week of dosing. Feed consumption (expressed as g/day) was decreased at 1000 mg/kg bw/day for study days (sd) 0-7. Feed consumption (expressed as g/kg bw/day) was reduced from sd 0-7 and increased for sd 7-14 at 1000 mg/kg bw/day. At necropsy of the F0 males, body weight was significantly reduced at 1000 mg/kg bw/day. In the F0 females, only body weight change was decreased at 1000 mg/kg bw/day for sd 0-7 but was similar to controls alter the 2-week prebreed period. Weight change was also reduced for gd 0-7 but not for the entire gestational period. Feed consumption (expressed as g/day and g/kg bw/day) was reduced at 1000 mg/kg bw/day) from sd 0-7. No effect of treatment was seen at any dose for the F0 female gestation and lactation periods. There was no evidence of reproductive toxicity in the F0 females at any dose level or any toxicity in the F1 offspring observed postnatally. At necropsy, there was no effect of treatment for absolute or relative organ weights. In conclusion, the administration of the test item by gavage once daily at 0, 50, 250, and 1000 mg/kg bw/day to rats, resulted in adult F0 parental toxicity at 1000 mg/kg bw/day but no indication of reproductive or developmental toxicity. Therefore, the F0 male and female systemic NOAEL was 250 mg/kg bw/day. The NOAEL for F0 reproductive toxicity and F1 offspring toxicity was greater than 1000 mg/kg bw/day.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Reliable without restrictions (guideline study)
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Male and female Sprague-Dawley rats were administered 1-chloro-2,3-dimethylbenzene orally by gavage at 0, 50, 250, and 1000 mg/kg bw/day for 2 weeks of prebreed exposure. Afterwards, the animals were randomly mated within treatment groups for a 2-week mating period to reproduce. Both sexes were exposed continuously through breeding and the females through 3 weeks of gestation and 4 days of lactation.

One female at 1000 mg/kg bw/day experiencing dystocia was euthanized moribund on gestational day (gd) 22; no other deaths occurred during conduct of the study. No other clinical signs were considered to be related to treatment. Body weights and weight change for the F0 males were decreased at 1000 mg/kg bw/day throughout the study although weight gain was similar to controls after the first week of dosing. Feed consumption (expressed as g/day) was decreased at 1000 mg/kg bw/day for study days (sd) 0-7. Feed consumption (expressed as g/kg bw/day) was reduced from sd 0-7 and increased for sd 7-14 at 1000 mg/kg bw/day. At necropsy of the F0 males, body weight was significantly reduced at 1000 mg/kg bw/day. In the F0 females, only body weight change was decreased at 1000 mg/kg bw/day for sd 0-7 but was similar to controls alter the 2-week prebreed period. Weight change was also reduced for gd 0-7 but not for the entire gestational period. Feed consumption (expressed as g/day and g/kg bw/day) was reduced at 1000 mg/kg bw/day) from sd 0-7. No effect of treatment was seen at any dose for the F0 female gestation and lactation periods. There was no evidence of reproductive toxicity in the F0 females at any dose level or any toxicity in the F1 offspring observed postnatally. At necropsy, there was no effect of treatment for absolute or relative organ weights. In conclusion, the administration of the test item by gavage once daily at 0, 50, 250, and 1000 mg/kg bw/day to rats, resulted in adult F0 parental toxicity at 1000 mg/kg bw/day but no indication of reproductive or developmental toxicity. Therefore, the F0 male and female systemic NOAEL was 250 mg/kg bw/day. The NOAEL for F0 reproductive toxicity and F1 offspring toxicity was greater than 1000 mg/kg bw/day.

Effects on developmental toxicity

Description of key information

Not yet available (testing proposal)

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available (further information necessary)
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

There is no evidence for species specific effects of the substance. Therefore the results of these in vivo data are regarded as relevant for humans.

Justification for classification or non-classification

With reference to the reliable reproductive/developmental toxicity screening study according OECD 421 resulting in the lack of reproductive/developmental effects, it is concluded that 1-chloro-2,3-dimethylbenzene is not subject to classification and labelling according to Regulation (EC) No 1272/2008 regarding reproductive and developmental toxicity.