Diphenylacetonitrile

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from read across substance.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Benzene-1,3-dicarbonitrile

EC Number:

210-933-7

EC Name:

Benzene-1,3-dicarbonitrile

Cas Number:

626-17-5

Molecular formula:

C8H4N2

IUPAC Name:

Benzene-1,3-dicarbonitrile

Test material form:

solid: particulate/powder

Remarks:

White powder

Details on test material:

The chemical was purchased from Tokyo Chemical Industry Co., Ltd. Purity was 99.9 and it was stored in a dark cool place (2-6 degree C) under a dense plug.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

The average body weights of male and female rats at the time of administration were 386 and 239 grams, respectively. The animals were placed individually in stainless steel write mesh cages (temperature 22 +/- 3 degree C; humidity of 55 +/-10%; 12/12 hour light/dark cycle). Nesting material was provided at day 0 of pregnancy. Water and food were available ad lib.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: Aqueous solution (1% methylcellulose)

Details on mating procedure:

No details given.

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

For male rats, the chemical was given two weeks before mating and thereafter for a total of 42 days of dosing. For female rats, the chemical was given two weeks before mating and thereafter until day 4 after delivery, for a total of 42-56 days of dosing.

Frequency of treatment:

Once per day

Doses / concentrations

Remarks:

0 (vehicle control), 6, 25 and 100 mg/kg bw/day

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:

The animals were examined for mortality and clinical signs twice per day. The body weight and food intake of each male rat were measured weekly. For female rats, the body weight and food intake of each animal were measured weekly before mating; on day 1, 7, 14, 20 of gestation; and on day 0 and 4 of lactation. Female rats were examined for oestrous cycle length, number of female rats with successful copulation, paring days until copulation, number of pregnant female rats, fertility index, number of corpora lutea, number of implantation sites, implantation index, number of female rats with parturition, gestation length, gestation index and number of female rats with live pups at day 4.

Oestrous cyclicity (parental animals):

Yes

Sperm parameters (parental animals):

Morphological evaluation of the spermatogenesis was performed post-mortem.

Litter observations:

Specific observations for the developmental phase of the study included number of pups born, delivery index, number of pups alive at day 0 of lactation, live birth index, sex ratio, number of pups alive on day 4 of lactation, viability index, and body weight of live pups at day 0 and 4 of lactation.

Postmortem examinations (parental animals):

All adult male and female rats were sacrificed after treatment for gross pathology, organ weight determinations and histopathology, which included a morphological evaluation of the spermatogenesis in the male rats.

Postmortem examinations (offspring):

All pups were examined externally for malformations and internally for visceral variations/malformations.

Statistics:

Parametric data: For multiple comparisons, Bartlett's variance test, Dunnett's test.
Non-parametric data: Kruskal Wallis.
Categorical data: Fischer test.
Histopathological data: Mann-Whitney

Reproductive indices:

Fertility index, implantation index, gestation index, and delivery index.

Offspring viability indices:

Live birth index and viability index.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical findings included excessive salivation in male and female rats treated at 100 mg/kg.

Mortality:

no mortality observed

Description (incidence):

All animals survived to planned death.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Male rats treated at 100 mg/kg showed significantly lower body weights on day 7, 14 and 28 of dosing (mean 8.1% lower) and decreased body weight gain between day 1 and 42 of dosing (by 16.4%) when compared to the control group. Female rats treated at 100 mg/kg showed lower body weights at day 14 and 20 of gestation (mean, 9.5%), markedly decreased body weight gain between day 0 to 20 of gestation (by 22.8%), and lower body weights at day 0 and 4 of lactation (mean, 11.3%) when compared to the control group.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Deviations in food intake in male rats were minor and occurred sporadically. In female rats, marked decreases in food intake were observed in the group of animals treated at 100 mg/kg on day 0 of lactation (56% lower) and day 3 of lactation (40% lower) when compared to the control group.

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, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Males: Slight hepatocellular hypertrophy at 100 mg/kg, a dose-dependent increase of hyaline droplets in the proximal tubular epithelium at ≥25 mg/kg and an increased tendency of tubule basophilia (indicative of renal toxicity) at 100 mg/kg. Microscopic examination of the male reproductive organs showed no treatment-related effects.
Females: Slight hepatocellular hypertrophy at 100 mg/kg. Microscopic examination of the female reproductive organs showed no treatment-related effects.

Histopathological findings: neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

No treatment-related effects observed.

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

The results from the spermatogenesis cycle test showed no significant effects attributed to the test chemical.

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no significant effects on oestrous cycle length, number of female rats with successful copulation, paring days until copulation, number of pregnant female rats, fertility index, number of corpora lutea, number of implantation sites, implantation index, number of female rats with parturition, gestation length, gestation index, or the number of female rats with live pups at day 4.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

histopathology: non-neoplastic

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

2

Dose descriptor:

LOAEL

Effect level:

<= 100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

body weight and weight gain

food consumption and compound intake

Results: F1 generation

General toxicity (F1)

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

The results from the developmental phase of the study showed a marked decrease in the number of live pups at day 0 and 4 of lactation at 100 mg/kg compared to the control group. This effect was accompanied by a marked decrease in the viability index at 100 mg/kg (mean viability, 28.0%) compared to the control group (mean viability, 97.5%).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean body weights of live pups were significantly lower on day 0 and 4 of lactation at 100 mg/kg compared to the control group.

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

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Gross examination revealed a low incidence of malformations at 0 mg/kg (0.6%) and 6 mg/kg (0.6%). No gross malformations were observed at 25 or 100 mg/kg. No visceral malformations were observed at any dose level. The incidences of visceral variants, which included persistent left umbilical artery and dilatation of renal pelvis, did not differ significantly between the groups.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

1

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

mortality

body weight and weight gain

other: Gorss and visceral malformations/variations

2

Dose descriptor:

LOAEL

Generation:

F1

Effect level:

<= 100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

mortality

body weight and weight gain

Overall reproductive toxicity

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

NOAEL for systemic toxicity in male and female rats was found at ≥25 mg/kg bw/day. NOAEL for toxicity to reproduction in both male and female rats was found at ≥100 mg/kg bw/day. The observed effects on development at 100 mg/kg bw/day were not considered to be solely non-specific, secondary to maternal toxicity.

Executive summary:

The chemical was given by oral gavage to 12 rats per sex per dose level at 0, 6, 25 and 100 mg/kg bw/day. For male rats, the chemical was given two weeks before mating and thereafter for a total of 42 days of dosing. For female rats, the chemical was given two weeks before mating and thereafter until day 4 after delivery, for a total of 42-56 days of dosing. Dose levels were selected based on the results from a dose range-finding study in which male and female rats were treated by oral gavage at 0, 50, 100, 200 and 400 mg/kg bw/day for a total of 14 days. The animals were examined for mortality and clinical signs twice per day. The body weight and food intake of each male rat were measured weekly. For female rats, the body weight and food intake of each animal were measured weekly before mating; on day 1, 7, 14, 20 of gestation; and on day 0 and 4 of lactation. Female rats were examined for oestrous cycle length, number of female rats with successful copulation, paring days until copulation, number of pregnant female rats, fertility index, number of corpora lutea, number of implantation sites, implantation index, number of female rats with parturition, gestation length, gestation index and number of female rats with live pups at day 4. Specific observations for the developmental phase of the study included number of pups born, delivery index, number of pups alive at day 0 of lactation, live birth index, sex ratio, number of pups alive on day 4 of lactation, viability index, and body weight of live pups at day 0 and 4 of lactation. All adult male and female rats were sacrificed after treatment for gross pathology, organ weight determinations and histopathology, which included a morphological evaluation of the spermatogenesis in the male rats. All pups were examined externally for malformations and internally for visceral variations/malformations. No mortality was observed during the dosing period. Clinical findings included excessive salivation in male and female rats treated at 100 mg/kg. Male rats treated at 100 mg/kg showed significantly lower body weights on day 7, 14 and 28 of dosing (mean 8.1% lower) and decreased body weight gain between day 1 and 42 of dosing (by 16.4%) when compared to the control group. Female rats treated at 100 mg/kg showed lower body weights at day 14 and 20 of gestation (mean, 9.5%), markedly decreased body weight gain between day 0 to 20 of gestation (by 22.8%), and lower body weights at day 0 and 4 of lactation (mean, 11.3%) when compared to the control group. Deviations in food intake in male rats were minor and occurred sporadically. In female rats, marked decreases in food intake were observed in the group of animals treated at 100 mg/kg on day 0 of lactation (56% lower) and day 3 of lactation (40% lower) when compared to the control group. There were no significant effects on oestrous cycle length, number of female rats with successful copulation, paring days until copulation, number of pregnant female rats, fertility index, number of corpora lutea, number of implantation sites, implantation index, number of female rats with parturition, gestation length, gestation index, or the number of female rats with live pups at day 4. At necropsy, male rats showed increased liver weights at ≥25 mg/kg, increased spleen weights at 100 mg/kg, decreased epididymides weights at 100 mg/kg and increased kidney weights at 100 mg/kg relative to the control data. These effects were accompanied by slight hepatocellular hypertrophy at 100 mg/kg, a dose-dependent increase of hyaline droplets in the proximal tubular epithelium at ≥25 mg/kg and an increased tendency of tubule basophilia (indicative of renal toxicity) at 100 mg/kg. Microscopic examination of the male reproductive organs showed no treatment-related effects. Furthermore, the results from the spermatogenesis cycle test showed no significant effects attributed to the test chemical. Female rats showed decreased thymus weights at ≥25 mg/kg, increased relative liver weights at ≥25 mg/kg and increased relative kidney weight at 100 mg/kg relative to the control data. These effects were accompanied by slight hepatocellular hypertrophy at 100 mg/kg. Microscopic examination of the female reproductive organs showed no treatment-related effects. The results from the developmental phase of the study showed a marked decrease in the number of live pups at day 0 and 4 of lactation at 100 mg/kg compared to the control group. This effect was accompanied by a marked decrease in the viability index at 100 mg/kg (mean viability, 28.0%) compared to the control group (mean viability, 97.5%). The mean body weights of live pups were also significantly lower on day 0 and 4 of lactation at 100 mg/kg compared to the control group. Gross examination revealed a low incidence of malformations at 0 mg/kg (0.6%) and 6 mg/kg (0.6%). No gross malformations were observed at 25 or 100 mg/kg. No visceral malformations were observed at any dose level. The incidences of visceral variants, which included persistent left umbilical artery and dilatation of renal pelvis, did not differ significantly between the groups. NOAEL for systemic toxicity in male and female rats was found at ≥25 mg/kg bw/day. NOAEL for toxicity to reproduction in both male and female rats was found at ≥100 mg/kg bw/day. The observed effects on development at 100 mg/kg bw/day were not considered to be solely non-specific, secondary to maternal toxicity.