4-chlorophthalic anhydride

Toxicity to reproduction

Administrative data

Endpoint:

one-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

1995

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4-chlorophthalic anhydride

EC Number:

204-251-9

EC Name:

4-chlorophthalic anhydride

Cas Number:

118-45-6

Molecular formula:

C8-H3-Cl-O3

IUPAC Name:

5-chloro-1,3-dihydro-2-benzofuran-1,3-dione

Test animals

Species:

rat

Strain:

other: Crl:CD (SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

Forty three male and 43 female Crl:CD(SD)IGS BR rats were received in good health from Charles River Laboratories, Inc., Raleigh, North Carolina. The animals were approximately 63 days old at receipt (weighing approximately 201 to 300 g). The females were nulliparous and nonpregnant. All animals were quarantined for approximately one week, during which time they were weighed, examined by a veterinarian and evaluated for ecto- and endo-parasites, clinical signs and acclimation to husbandry. All animals were considered to be in good health and suitable for use in this study. There were 80 animals (40 males and 40 females) assigned to the study during the quarantine period. All animals were uniquely identified prior to initiation of the study by eartag. The weight variation of the study animals at initiation did not exceed +/- 20% of the mean weight for each sex. The animals were individually housed during the quarantine period and upon initiation of the treatment period in solid-bottom polycarbonate cages with stainless steel wire lids with chip cage litter. Animals were housed 2 per cage during the mating period. Females were caged individually once they were successfully mated (or at the end of the mating period) and throughout gestation. Females were housed with their litters throughout the lactation period. All animal rooms were on a 12 hour light cycle per day and were air-conditioned. Temperature and relative humidity (RH) were continuously monitored, controlled and recorded. Temperature and RH readings over the course of the study were 70.6-75.2°F and 42.6-62.3%, respectively. The basal diet and city tap water were provided ad libitum.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Test article formulations were prepared at 10, 50 and 250 mg/kg of 4-CLPA in corn oil at 5 ml/kg. The appropriate aliquot of 4-CLPA was weighed into a beaker and a portion of the corn oil was added and stirred to mix. The remaining corn oil was added to the calibration mark. The formulations were stirred for 15 minutes and two 20-mL samples were collected as an analytical sample and an archived sample. The bulk sample was transferred to amber bottles for dosing. The doses were formulated twice during the study and analyzed.

Details on mating procedure:

Following the 14-day prebreeding period, the animals were mated based on 1 male to 1 female, selected randomly within each dose group for a period of 14 days, with no change in mating partners. The observation of vaginal sperm or copulation plug was considered evidence of successful mating. Females were examined daily during the cohabitation period for the presence of sperm or copulation plug in the vaginal tract. The day vaginal sperm (or plug) were observed was designated gestation day (gd) 0. Once vaginal sperm were observed, the male and female from that mating pair were individually housed. 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:

Two 20-mL samples were collected from each dose group as an analytical sample and an archived sample. Triplicate samples (1 mL each) were collected from the analytical sample of each dose formulation for analysis by high performance liquid chromatography (HPLC). Dose formulations of 4-CLPA prepared in corn oil at 10, 50 and 250 mg/kg and used in animal studies were found to contain between 98.3 and 101% of the nominal concentration of 4 CLPA. The relative standard deviations for triplicate sample analyses were less than or equal to 1.5%. The dose formulations of 4-CLPA had been previously demonstrated to be homogeneous and stable for at least 35 days under ambient storage conditions.

Duration of treatment / exposure:

Males: Two weeks prior to mating, during the two week mating period
Females: Two weeks prior to mating, during the two week mating period, during gestation (up to three weeks) and through lactation day 4

Frequency of treatment:

Daily

Details on study schedule:

The two week pre-breeding treatment began for all F0 animals when the animals were approximately 10 weeks old. The animals were approximately 12 weeks old when mated for two weeks.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

10 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

250 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

The doses were chosen based on a 28-day repeat dose toxicity study which was conducted with 4-CLPA at doses of 0, 10, 100, and 300 mg/kg/day administered by oral gavage for 28 consecutive days. At 300 mg/kg/day, there was weight loss (predominantly in males), clinical signs and lesions of the stomach. In addition, a functional observational battery performed weekly and motor activity performed near the end of the study indicated that 4-CLPA did not cause any neurotoxic effects at any dose. Therefore, based on these observations, the doses chosen for the subject study were 0, 10, 50, and 250 mg/kg/day.

Positive control:

not required

Examinations

Parental animals: Observations and examinations:

Observations for mortality were made twice daily (a.m. and p.m.), and the general condition of all animals was checked daily. Clinical examinations were conducted and recorded daily throughout the course of the study. This record included the day of onset, degree, and duration of symptoms. These cageside 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. The body weights of the F0 male rats were determined and recorded initially and then weekly throughout mating. The body weights of F0 female rats were recorded in the same manner until confirmation of mating. During gestation, F0 females were weighed on gd 0, 7, 14, and 20. Dams producing litters were weighed on lactational days (pnd) 0 and 4, and body weight gains were computed. Feed consumption measurements were recorded weekly for all F0 parental study animals during the prebreed treatment period. During pregnancy of F0 females, feed consumption was recorded for gd 0-7, 7-14, and 14-20. During lactation of the F1 litters, maternal feed consumption was measured for pnd 0-4. Feed consumption was not measured during the cohabitation period since 2 adult animals (breeding pair) were in the same cage. Feed consumption collection periods corresponded to the collection of the animals’ body weight data. Beginning on gd 20, each female was observed twice daily for evidence of littering.

Sperm parameters (parental animals):

Ten high dose and control F0 males were examined at necropsy with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.

Litter observations:

All live F1 pups were counted, sexed, weighed, and examined as soon as possible after birth (date of birth designated pnd 0) to determine the number of viable pups from each litter. All stillborn pups or pups that died on the date of birth were sexed and counted. Thereafter, litters were evaluated daily for survival through pnd 4. The body weights and sexes were recorded on an individual basis, but the pups were not uniquely identified. All pups were examined for physical abnormalities at birth and through pnd 4. All pups dying during lactation were necropsied, when possible, to investigate the cause of death.

Postmortem examinations (parental animals):

All F0 parental animals were subjected to a complete gross necropsy, with selected organs (see below) weighed. Uterine nidation scars and total number of corpora lutea were recorded for F0 females. The following organs were weighed and retained in neutral buffered 10% formalin from all F0 adult animals (except testes and epididymis, which were retained in Bouin’s fixative for 24 hours and then stored in 70% ethanol): epididymides (pair); ovaries (pair); prostate; seminal vesicles with coagulating glands and their fluids (pair); testes (pair) and uterus with cervix and vagina. In addition to the organs listed above, all gross lesions were retained in neutral buffered 10% formalin. Full histopathology of the organs listed above was performed for the 10 high dose and control F0 males and females (with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure). Scheduled sacrifice of the F0 parental males occurred after the 28-day dosing period, and scheduled sacrifice of the F0 maternal animals occurred on pnd 4. All females except one animal in the 50 mg/kg/day group were pregnant. Organ weights were reported as absolute and relative to terminal body weight.

Postmortem examinations (offspring):

All pups dying during lactation were necropsied, when possible, to investigate the cause of death.

Statistics:

The unit of comparison was the male, female, pregnant female, or the litter, as appropriate. Treatment groups were compared to the concurrent control group using either parametric ANOVA or robust regression methods. If Levene’s Test (homogeneity of variance assumption) indicated lack of homogeneity of variance, robust regression methods were used to test for overall treatment group differences, followed by Wald Chi-Square Tests for exposed vs. control group comparisons, if the overall treatment effect was significant. If Levene’s Test did not reject the hypothesis of homogeneous variances, standard ANOVA techniques were applied for comparing the treatment groups and, when significant, to compare each exposed group to the control via Dunnett’s Test. For the litter-derived percentage data, the ANOVA was weighted according to litter size. A one-tailed 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. 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, with body weight at acquisition or measurement as the covariate. For correlated data, analysis of overall significance, presence of trend, and pairwise comparisons to the control group values was determined. A test for statistical outliers was performed on parental body weights, feed consumption (in g/day), and organ weights. The data excluded from summarization and analysis and were designated as outliers. For all statistical tests, p is less than or equal to 0.05 was used as the criteria for significance.

Reproductive indices:

The indices for reproductive performance and gestational parameters (%) that were calculated for this study were as follows: mating and fertility index for males and females; gestational index for females; and pregnancy index for males. The following endpoints were also calculated for each litter (dam): percent postimplantation loss per dam and arcsine root transformation; stillbirth index per dam and arcsine root transformation; live birth index per dam and arcsine root transformation; four-day survival index per dam and arcsine root transformation; percent males per litter and arcsine root transformation; average pup weight per litter; average male pup body weight per litter; and average female pup body weight per litter.

Offspring viability indices:

The indices for postnatal parameters (%) that were calculated for this study are as follows: live birth index and 4-day survival index.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

F0 MALES
- Treatment-related observations included 6 males at 250 mg/kg/day and 1 male at 50 mg/kg/day with audible respiration; 1 male at 250 mg/kg/day gasping postdosing; and 1 male at 250 mg/kg/day sneezing prior to dosing. Other findings were not considered treatment-related except for a high incidence of postdose rooting, which 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.

F0 FEMALES
- Clinical observations recorded included audible respiration in 5 females at 250 mg/kg/day after dosing, and 1 female each at 250 mg/kg/day was gasping, had labored breathing, sneezing, and salivation. One female at 250 mg/kg/day was emaciated. Other findings were not considered treatment-related except for a high incidence of postdose rooting and struggling during dosing and salivation, which 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.
- Treatment-related clinical observations included 0, 1, 1, and 4 females with audible respiration at 0, 10, 50, and 250 mg/kg/day; 1 female with pica at 250 mg/kg/day; 1 female gasping at 250 mg/kg/day, 2 females gasping postdosing at 250 mg/kg/day, 1 female gasping postdosing at 10 mg/kg/day, and 1 female gasping prior to dosing at 250 mg/kg/day. One female was sneezing at 250 mg/kg/day, and 1 female was sneezing postdosing at 10 mg/kg/day. Other clinical signs were not considered treatment-related except for signs of taste aversion (described above), which are not toxic signs.
- There were no significant differences in F0 maternal body weights or feed consumption (expressed as g/day or g/kg/day) during lactation at any dose. Treatment-related clinical observations included 0, 0, 1, and 6 females with audible respiration at 0, 10, 50, and 250 mg/kg/day; 1 female gasping at 250 mg/kg/day, and 2 females gasping prior to dosing at 250 mg/kg/day. Four females were sneezing and 2 females were emaciated at 250 mg/kg/day. Other clinical signs were not considered treatment-related except for signs of taste aversion (described above), which are not toxic signs.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

F0 MALES
- No unscheduled deaths occurred in the F0 males.

F0 FEMALES
- One unscheduled F0 female death occurred on study day (sd) 40 (pnd 2) at 250 mg/kg/day, and the cause of death could not be determined although a small amount of oil was observed throughout the viscera suggesting the possibility of perforation of the esophagus or stomach during dosing. Another F0 female at 250 mg/kg/day was euthanized moribund on sd 42 (pnd 2) after experiencing signs of respiratory distress.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

F0 MALES
- There were no significant effects of treatment on F0 male body weight or body weight change. However, body weight gain in the 250 mg/kg/day group was approximately 20% less than the control group for the 28-day dosing period indicating that 4-CLPA had a slight effect on male body weight at this dose.
- At scheduled sacrifice, mean body weight was unaffected across dose groups.

F0 FEMALES
- There were no significant differences among groups for F0 female body weights during the prebreed or mating (sd 0-28) periods, but there was a decrease in body weight change from sd 0 to 7 at 250 mg/kg/day during the prebreed period.
- There were no significant differences in the F0 maternal body weights or body weight change during gestation.
- There were no significant differences in F0 maternal body weights or feed consumption (expressed as g/day or g/kg/day) during lactation at any dose.
- At scheduled sacrifice, mean body weights of F0 females were equivalent across all dose groups.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

F0 MALES
There were no differences in feed consumption across dose groups for F0 males.

F0 FEMALES
Feed consumption (expressed as g/day or g/kg/day), was significantly reduced at 250 mg/kg/day for sd 0 to 7, 10 and 50 mg/kg/day for sd 7 to 14 (g/day only), and at 50 and 250 mg/kg/day for sd 0 to 14. The changes in feed consumption, other than for the high dose group, were considered unrelated to toxicity of the test substance due to lack of a dose-response, lack of associated change in body weight, and/or consistent pattern of effect. There were no significant differences in the F0 maternalfeed consumption during gestation.

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

F0 MALES
- Other than hyperplasia of the forestomach and inflammation of the glandular stomach in animals with grossly observed lesions (three males from the high dose group), there were no treatment-related microscopic findings.

F0 FEMALES
- Other than stomach lining sloughing (3 females from the high dose group) in animals with grossly observed lesions, there were no treatment-related microscopic necropsy findings of F0 parental females.

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

During the postmating period (sd 28 to 35), there were 2 females at 250 mg/kg/day that were sperm-negative. However, only 1 female at 50 mg/kg/day was not pregnant, and 1 pregnant female at 10 mg/kg/day did not deliver a litter.

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no significant effects of exposure to the test substance on F0 mating, fertility, or pregnancy indices during the production of F1 offspring, 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).

Details on results (P0)

No unscheduled deaths occurred in the F0 males. Therefore, there were 10, 10, 10, and 10 F0 males that were evaluated at scheduled sacrifice at 0, 10, 50, and 250 mg/kg/day, respectively. One unscheduled F0 female death occurred on study day (sd) 40 (pnd 2) at 250 mg/kg/day, and the cause of death could not be determined although a small amount of oil was observed throughout the viscera suggesting the possibility of perforation of the esophagus or stomach during dosing. Another F0 female at 250 mg/kg/day was euthanized moribund on sd 42 (pnd 2) after experiencing signs of respiratory distress. Therefore, there were 10, 10, 10, and 8 F0 females evaluated at scheduled sacrifice at 0, 10, 50, and 250 mg/kg/day, respectively.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

gross pathology

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

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 at 0, 10, 50, and 250 mg/kg/day, 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 mortality for pnd 0-4 was 5, 7, 2, and 14 at 0, 10, 50, and 250 mg/kg/day, respectively. Of the 14 pups that died in the 250 mg/kg/day group, 12 were from one litter. In addition, the pups from the two dams that died on pnd 2 were either found dead or were sacrificed on or near pnd 2. These pups were not considered to be found dead for determination of survival. Thus a total of 28 pups were found dead (including five pups from pnd 0 not considered in the “live” count for survival) and 20 pups were sacrificed from the 250 mg/kg/day group.

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.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Necropsy findings of F1 pups found dead or euthanized moribund on pnd 0-4 included pups that died on pnd 0, 1, and 2 exhibiting open (fetal state) or closed (postnatal state) ductus arteriosis, no air (fetal state) or air (postnatal state) in lungs, no or little milk in the stomach, anal atresia, and autolysis. The most common finding was no milk in the stomach, indicating that they were either not nursing or not able to receive milk through nursing.

Details on results (F1)

There were 10, 9, 9, and 10 live litters on pnd 0, and 10, 9, 9, and 7 live litters on pnd 4, at 0, 10, 50 and 250 mg/kg/day, respectively. The reduction in the number of live litters at 250 mg/kg/day was the result of the death of 2 dams (1 found dead and 1 sacrificed moribund), and one litter in which all pups were either dead or missing on pnd 2.

The sex ratio (percent male pups/litter) was unaffected at any dose for any interval.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

> 250 other: mg/kg

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no treatment-related findings observed

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Summary and Statistical Analysis of Select F0 Male Body Weight

Change During the Prebreed and Mating Periods

	4-CLPA (mg/kg/day)
Group:	0	10	50	250
Body Weight Change (g)
SD 0 to 28
Mean	109.8	110.2	117.3	88.9
S.E.M.	7.0	7.2	10.8	5.9
N	10	10	10	10
S.E.M. = Standard error of the mean SD = Study day N = Number of animals

Summary and Statistical Analysis of Select F0 Female Body Weight

Change During the Prebreed, Mating and Postmating Holding Periods

	4-CLPA (mg/kg/day)
Group:	0	10	50	250
Body Weight Change (g)
SD 0 to 7
Mean	18.2	13.8	10.9*	11.2*
S.E.M.	2.6	1.5	1.6	2.0
N	10	10	10	10
* = p is less than 0.05; Dunnett’s test S.E.M. = Standard error of the mean SD = Study day N = Number of animals

Summary and Statistical Analysis of Select F0 Female Feed

Consumption During the Prebreed Period

	4-CLPA (mg/kg/day)
Group:	0	10	50	250
Feed Consumption (g/day)
SD 0 to 7
Mean	17.6	16.9	16.1	15.2**
S.E.M.	0.6	0.4	0.4	0.5
N	10	10	10	10
SD 7 to 14
Mean	17.5	16.1F	15.6FF	16.7
S.E.M.	0.4	0.4	0.4	0.9
N	10	10	10	10
SD 0 to 14
Mean	17.6	16.5	15.9*	15.9*
S.E.M.	0.5	0.4	0.4	0.5
N	10	10	10	10
Feed Consumption (g/kg/day)
SD 0 to 7
Mean	71.4	69.8	66.4	53.4***
S.E.M.	1.5	2.1	2.3	2.0
N	10	10	10	10
SD 0 to 14
Mean	72.0	68.0	66.1*	66.1*
S.E.M.	1.2	1.7	0.9	1.6
N	10	10	10	10
* = p is less than 0.05; Dunnett’s test ** = p is less than 0.01; Dunnett’s test *** = p is less than 0.001; Dunnett’s test F = p is less than 0.05; Individual t-test for pairwise comparisons to control in robust regression model. FF = p is less than 0.01; Individual t-test for pairwise S.E.M. = Standard error of the mean SD = Study day N = Number of animals

Applicant's summary and conclusion

Conclusions:

The parental NOAEL was determined to be 100 mg/kg bw/day and the NOAEL for reproductive toxicity was determined to be >250 mg/kg bw/day.

Executive summary:

Toxicity to reproduction was determined in an OECD 421 study performed according to GLP criteria. In this study, 80 Crl:CD (SD) rats (40 males, 40 females) were exposed to the test substance in doses of 0, 10, 50 and 250 mg/kg bw/day. The doses chosen were based on a 28-day repeated dose toxicity study. Males were exposed two weeks prior to mating and during the two week mating period, whereas females were exposed two weeks prior to mating, during the two week mating period and during gestation (up to three weeks) and through lactation day 4. The test substance dissolved in corn oil was administered orally via gavage. Animals were observed twice daily to determine toxicity, and general condition including clinical signs was checked daily. Additionally, body weight, feed consumption, organ weights and gross necropsy were examined of the parental animals. Histopathology was performed on organs from the high dose group and the control. Emphasis was given to reproductive indices. Furthermore, pups that died during lactation were necropsied. Mating and fertility index for males and females; gestational index for females; and pregnancy index for males were also determined. In the F0 males, the only adverse effects were respiratory distress (considered to be related to the irritant properties of the test substance), a slight decrease in body weight gain, and lesions of the stomach at the high dose. In the F0 females, the only adverse effects were decreases in body weight change (during sd 0 to 7 of the prebreed period only), treatment-related clinical signs and lesions of the stomach (local effect) at the high dose. The relationship of the two deaths at the high dose on pnd 2 to 4-CLPA toxicity was uncertain. There was no other evidence of reproductive toxicity in the F0 females at any dose or toxicity in the F1 offspring observed postnatally. Therefore, the F0 male and female systemic NOAEL was 50 mg/kg/day. The NOAELs for F0 reproductive toxicity and F1 offspring toxicity were greater than 250 mg/kg/day.