Mixture of reaction products of substituted copper phthalocyanine, hydrogenated resin acids, alkaline earth metal chlorides, and copper phthalocyanine

Toxicity to reproduction

Administrative data

Endpoint:

toxicity to reproduction

Remarks:

other: screening reproduction/developmental

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April - June 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Performed according to OECD 421 and GLP principles.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: US EPA OPPTS 870.3550

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

B508

IUPAC Name:

B508

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material: B508
- Physical state: powder

Test animals

Species:

rat

Strain:

other: Crl: WI (Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 11 wks
- Weight at study initiation: 295-320 g for males; 182-208 g for females
- Housing: in Macrolon cages with sterilized sawdust as bedding material and paper as cage-enrichment; 5 animals/sex/cage pre-mating; 1 male and 1 female per cage during mating; at post-mating males were kept with a maximum of 5 animals/cage and females were individually housed.
- Diet: free access to pelleted rodent diet
- Water: free access to tap-water
- Acclimation period: at least 5 days prior to start of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.2-21.4
- Humidity (%): 33-76
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 29 April - 17 June 2009

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.5 w/v% methylcellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: prepared daily within 6 hours prior to dosing and homogenized to a visually acceptable level

VEHICLE
- Justification for use and choice of vehicle (if other than water): based on information provided by the sponsor and trial formulations at the test lab.
- Amount of vehicle (if gavage): 5 ml/kg bw

Details on mating procedure:

- M/F ratio per cage: 1/1 (one female was cohabitated with one male of the same treatment group, avoiding sibling mating)
- Length of cohabitation: maximum 14 days with one male
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after an unsuccessful attempt: no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentrations analysed at 50 and1000 mg/kg bw/d, based on copper and strontium analysis using ICP-MS, were in agreement with the target concentrations (i.e. mean accuracies between 85 and 115%). The concentrations analysed at 250 mg/kg bw/d, based on copper analysis, were slightly above target concentration (mean accuracy 116%) but the result was accepted since the corresponding result based on strontium analysis was well within the criterion range.

Duration of treatment / exposure:

Males were exposed for 28 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to necropsy. Females were exposed for 40 to 49 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 3 days of lactation.

Frequency of treatment:

Once daily for 7 days per week. Animals were dosed up to the day prior to scheduled necropsy.

Details on study schedule:

- Age at mating of the mated animals in the study: approximately 13 weeks

Doses / concentrations

Remarks:

Doses / Concentrations:
50, 250 and 1000 mg/kg bw/d
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: the selected dose levels are based on the outcome of the 28-day repeated dose study (CERI B11-0899; see 7.5.1). The NOAEL of this study was 1000 mg/kg bw/d based on the absence of effects.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum, and during lactation on Days 1 and 4.

FOOD CONSUMPTION: Yes
Weekly, for males and females. Food consumption was not recorded during the breeding period. Food consumption of mated females was measured on day 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on day 1 and 4.

WATER CONSUMPTION : No
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

Oestrous cyclicity (parental animals):

Not determined.

Sperm parameters (parental animals):

Parameters examined in male parental animals: weight of testes and epididymides of all males; staging of spermatogenesis in control males and at 1000 mg/kg bw/d

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, weight gain, general abnormalities.
Possible defects or cause of death were evaluated.

- Mortality: number of live and dead pups on day 1-4 of lactation
- Clinical signs: at least once daily, detailed clinical observations were made in all animals.
- Body weights: live pups were weighed on day 1 and 4 of lactation.
- Sex: Sex was determined for all pups on day 1 and 4 of lactation (by assessment of the ano-genital distance).

GROSS EXAMINATION OF DEAD PUPS:
yes, on postnatal day 4

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All animals following completion of the mating period (minimum of 28 days of dose administration)
- Maternal animals: All animals on lacatation day 4 or shortly thereafter or for females that failed to deliver on post-coitum day 25-27 (females with evidence of mating) or approximately 21 days after the last day of the mating period (females without evidence of mating).

GROSS NECROPSY
- Gross necropsy consisted of examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs.

HISTOPATHOLOGY / ORGAN WEIGHTS
Organ weights of epididymides and testes was determined.
The following slides were examined by a pathologist:
- Ovaries and testes of animals from control group and 1000 mg/kg bw/d group and the epididymides of all males
- Cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testes, uterus and vagina of all following animals:
Control: one male and one female who failed to mate
50 mg/kg bw/d: one male and one female (female was not pregnant)
250 mg/kg b/d: one male and one female (female was not pregnant)
1000 mg/kg bw/d: one male and one female who failed to mate
- All gross lesions of all animals

Postmortem examinations (offspring):

All offspring was sexed and externally examined. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated.

Statistics:

The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (Dunnett, 1955) (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (Miller, 1981) (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test (Fisher 1950) was applied to frequency data.
- The implantation index, number of live pups born, sex ratio and delivery index were subjected to the Kruskal-Wallis nonparametric ANOVA test (Kruskal, 1952) to determine intergroup difference. If the results of the ANOVA were significant (p<0.05), the Wilcoxon test (Wilcoxon, 1945) was applied to the data to compare the treated groups to the control group.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may be rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

No statistical analysis was performed on histopathology findings.

Reproductive indices:

percentage mating, fertility index, conception rate, implantation index, gestation index, duration of gestation

Offspring viability indices:

delivery index, percentage live males/females at first litter check, sex ratio, percentage of postnatal loss days 0-4 post partum, viability index

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

effects observed, treatment-related

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No mortality occurred. There were no treatment-related clinical signs of toxicological relevance.
Salivation was noted for the majority of all animals treated at 1000 mg/kg . Since salivation was only slight and occurred only intermittently during the study, this finding was considered to be of no toxicological significance.
It was noticed that the test substance is excreted via the feces. Due to the intense blue color of the test substance, blue discoloration of the feces was noted in all treated animals. Upon contact with the feces, also the tails of several animalsat 1000 mg/kg bw (both sexes) stained blue. These observations were considered to be an indirect effect of the test substance and not toxicologically relevant.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Body weights and body weight gain of treated animals remained in the same range as controls over the study period. No toxicologically significant changes were observed for food consumption before or after allowance for body weight up to 1000 mg/kg bw.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
The spermatogenic staging profiles were normal for all control and high dose males.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Breeding parameters were unaffected by treatment up to 1000 mg/kg bw/day. There were no findings in the reproductive organs of any male or female animals suspected of infertility that would account for poor reproductive performance.

ORGAN WEIGHTS (PARENTAL ANIMALS)
No effect on organ weights was noted.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Macroscopic observations at necropsy revealed a yellowish or greenish soft nodule(s) in the epididymides (tail) in one male at 50 mg/kg bw/day and two males at 1000 mg/kg bw/day.
The bluish discoloration of the tail and bluish contents noted in different parts of the gastro-intestinal tract was caused by the blue staining ability of the test substance. Therefore, this observation was not considered to be a sign of toxicity.
Incidental findings included tan focus in the preputial or clitoral glands, an uterus containing fluid, and alopecia, scabbing and sore(s) on different parts of the body. These findings are occasionally seen among rats used in these types of studies and/or in the absence of a dose response relationship they were considered to be changes of no toxicological significance.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Sperm granulomas were recorded at a slight degree in single males of controls, 50 mg/kg bw/day and 1000 mg/kg bw/day. A moderate degree of sperm granulomas was noted in one male at 50 mg/kg bw/day and three males at 1000 mg/kg bw/day. This finding was the histologic correlate to the greenish-yellowish soft nodules noted at necropsy. There were two cases of bilateral sperm granulomas in males at 1000 mg/kg bw/day. Although sperm granulomas are not unusual findings in rats fo this age and strain, the incidence and severity at 1000 mg/kg bw/d is somewhat higher than the historical control values, and therefore a relationship to treatment cannot be excluded. No further histopathological findings were noted.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

parental toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: increased incidence and severity of sperm granulomas at 1000 mg/kg bw/d

2

Dose descriptor:

NOAEL

Remarks:

reproduction toxicity

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed at highest dose tested

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Details on results (F1)

VIABILITY (OFFSPRING)
Postnatal loss and viability index were similar for the control and treated groups.

CLINICAL SIGNS (OFFSPRING)
Development of pups was unaffected by treatment up to 1000 mg/kg bw/day. Incidental clinical symptoms consisted of (very) small appearance, blue spots on several body parts and a wound and scabbing of the hindleg.

BODY WEIGHT (OFFSPRING)
(Mean) body weights were similar for the control and treated groups.

GROSS PATHOLOGY (OFFSPRING)
Macroscopic examination of the pups revealed small appearance, and autolysis and/or absence of milk for dead pups. No relationship with treatment was established for these observations and they were considered to be within the normal biological variation for rats of this age and strain.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

In a screening reproduction/developmental toxicity study in rats the NOAEL for parental toxicity was 250 mg/kg bw/d and the NOAEL for reproduction and developmental toxicity was established as being at least 1000 mg/kg bw/d.

Executive summary:

Rats were dose with 0, 50, 250 or 1000 mg/kg bw/d B508 via gavage in a screening reproduction/developmental toxicity study performed according to OECD 421 and GLP principles.

No mortality occurred and no clinical signs were observed. No effect on body weight, food consumption or organ weight was noted. No difference in spermatogenic staging profile was seen between treated and control groups. At macroscopy a yellowish or greenish soft nodule was noted in the epididymides (tail), which correlated with an increased incidence and severity of sperm granulomas observed at 1000 mg/kg bw/d leading to a parental NOAEL of 250 mg/kg bw/d.

Breeding parameters were unaffected by treatment up to 1000 mg/kg bw/day. There were no findings in the reproductive organs of any male or female animals suspected of infertility that would account for poor reproductive performance. Therefore, the NOAEL for reproduction toxicity was established to be at least 1000 mg/kg bw/d.