Benzoic acid, 4-[2-(2-hydroxy-6-sulfo-1-naphthalenyl)diazenyl]-, strontium salt (2:1)

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 Mar 2012 - 16 Oct 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

adopted 1995

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain as given in study report: RccHanTM: WIST(SPF)
- Source: Harlan Laboratories, Netherlands
- Age at study initiation: 11 weeks
- Weight at study initiation: Males, 294 to 420 g; Females, 206 to 240 g
- Housing: in groups of 3 to 4 (up to 6 during acclimatization) in Makrolon type-4 cages with wire mesh tops up to the day of randomization on day 6 of acclimatization; afterwards, individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding with paper enrichment.
- Diet: pelleted standard Harlan Teklad 2914C rodent maintenance diet (Provimi Kliba SA, Kaiseraugst, Switzerland, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 30 - 70
- Air changes (per hr): 10 to 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

purified, prepared in-house using an ELGA water purification system (Ultra Bio No. UBH 279651)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was weighed into a glass beaker on a tared precision balance and the vehicle was added and a homogeneous suspension prepared.
Separate formulations were prepared for each concentration. Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.
APPLICATION VOLUME:
10 mL/kg bw

Details on mating procedure:

For mating, the females were housed with sexually mature males from the same dose group at a 1:1 ratio until evidence of copulation was observed. Copulation was evidenced either by sperm positive analysis of daily vaginal smear or by the presence of a copulation plug.
The day on which a positive mating was determined (copulation plug or sperm) was designated day 0 post coitum (day 0 p.c.).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

On the first treatment day and the last week of the treatment samples from the control group (middle only) as well as 3 samples (top, middle and bottom) of each concentration were taken prior to dosing for analysis of concentration and homogeneity. The aliquots for analysis were frozen and delivered on dry ice to the sponsor for analysis. Analysis of the formulations was performed according to an established method and reported (BASF SE, Competence Center Analytics, Study No. 12L00250, 20 Aug 2012).

Duration of treatment / exposure:

Males: at least 4 weeks
Females: approximately 7 weeks

Frequency of treatment:

once daily

Details on study schedule:

First test item administration was done on day 1 of pre-pairing (males and females).
Pre-pairing duration was 14 days (males and females).
Pairing period did not exceed 14 days.
Gestation duration was approximately 21 days (females).
Treatment was terminated on day 3 post partum for the females, and one day before sacrifice for the males.
Females and pups were sacrificed for necropsy on day 4 post partum.
Males were sacrificed for necropsy after a treatment period of at least 28 days (or more if necessary for assessing reproductive effects).

Remarks:

Doses / Concentrations:
100, 300, 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

11

Control animals:

yes, concurrent no treatment

Parental animals: Observations and examinations:

CLINICAL SIGNS AND MORTALITY
The animals were checked twice daily for mortality.
Cage-side clinical observations were made once daily; additionally females were observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.

BODY WEIGHT
Body weights were recorded daily from treatment start to day of necropsy.

FOOD CONSUMPTION
For the males, food consumption was recorded for the following periods:
- pre-pairing period days 1 - 4, 4 - 8, 8 - 11 and 11 - 14;
- after pairing period weekly.
For the females, food consumption was recorded for the following periods:
- pre-pairing period days 1 - 4, 4 - 8, 8 - 11 and 11 - 14;
- gestation days 0 – 7, 7 - 14 and 14 – 21;
- lactation days 1 - 4.
No food consumption was recorded during the pairing period.

WATER CONSUMPTION
Not considered.

NECROPSY
At test ending, all animals were sacrificed (injection of sodium pentobarbital) and exsanguinated.

Litter observations:

The litters were examined for litter size, live births, still births and any gross anomalies.
The sex ratio of the pups was recorded.
Pups were weighed individually on day of parturition and on day 1 and 4 post partum.

Postmortem examinations (parental animals):

GROSS PATHOLOGY
All parent animals were subjected to gross pathological examination, either at the scheduled necropsy or during the study if death occurred.
Particular attention was given to the organs of the reproductive system.
The number of implantation sites and corpora lutea was recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites.

ORGAN WEIGHING
Organs were weighed; especially the testes and epididymides of all parental males were weighed separately.

TISSUE PREPARATION
The ovaries (including oviduct) and uterus (including cervix and vagina) from all parental females were preserved in neutral phosphate buffered 4% formaldehyde solution. The testes and epididymides from all parental males were preserved in Bouin’s fixative. The prostate and seminal vesicles (including coagulating gland) from all males were fixed in neutral phosphate buffered 4% formaldehyde solution. Gross lesions were preserved in neutral phosphate buffered 4% formaldehyde solution.

HISTOPATHOLOGY
All organ and tissue samples to be examined were processed, embedded and sectioned; the 2 to 4 micrometers thick sections were stained with hematoxylin and eosin. Additionally, the testis was stained by PAS-hematoxylin.
Slides of testes, epididymides and ovaries from all animals of the control and high-dose groups were examined, as well as all occurring gross lesions. Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure. Histological examination of ovaries was carried out on any females that did not give birth. In addition, microscopic examination of the reproductive organs of all infertile males was made, if necessary.

Postmortem examinations (offspring):

Dead pups, except those excessively cannibalized, were subjected to gross pathological examination. In fact, all pups were examined for any structural changes, either at the scheduled necropsy or during the study (day 1 to 4 post partum) if death occurred.

Statistics:

The following statistical methods were used to analyze food consumption, body weights, organ weights and reproduction data:
Means and standard deviations of various data, the Dunnett-test (many to one t-test; Dunnett CW, J. Amer. Statist. Assoc. 50: 1096-1121, 1955) ) based on a pooled variance estimate, if the variables were assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex, the Steel-test (many-one rank test; Miller RG, Simultaneous Statistical Inference, Springer Verlag, New York, 1981) instead of the Dunnett-test when the data could not be assumed to follow a normal distribution, and Fisher's exact-test (Fisher RA, Statistical Methods for Research Workers, Oliver and Boyd, Edinburgh, 1950) for variables that could be dichotomized without loss of information.

Reproductive indices:

Fertility index (number of females achieving pregnancy as % of females paired), conception rate ((number of females achieving pregnancy as % of females mated), and gestation index (number of females with living pups as % of females pregnant) were determined.

Offspring viability indices:

Birth index (number of pups born alive as % of implantation).

CLINICAL SIGNS AND MORTALITY
All animals survived the scheduled study period. Stained/discolored red feces were recorded in most or all test item treated animals of both genders from the start of dosing until necropsy. Severity increased with increasing dose levels. No further clinical signs were noted at any dose level.
One female treated at 100 mg/kg bw/day was reported to be in a weakened condition on the first day of the lactation period. This was accompanied by hunched posture until day 2 post partum and slightly ruffled fur until the scheduled necropsy. This animal lost its whole litter. This case was incidental and since no dose-dependency could be evidenced, it was not regarded at test item-related.

BODY WEIGHT
For the parental males, there were no effects on mean body weights and mean body weight gain at any dose level. The overall values of mean body weight gain at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day were: +8%, +8%, +8% and +9% during the pre-pairing period, +2%, +3%, +2% and +3% during the pairing period and +4%, +5%, +4% and +5% during the after pairing period (percentages refer to the body weight gain within the period).
For the parental females, there were no effects on mean body weights and mean body weight gain at any dose level and any study phase. The overall values of mean body weight gain at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day were: +6%, +6%, +7% and +7% during the pre-pairing period, +50%, +50%, +46% and +51% during the gestation period and +4%, +7%, +7% and +7% during the lactation period (% refers to body weight gain within the period).

FOOD CONSUMPTION
For the parental males, there were no effects on mean food consumption at any dose level.
For the parental females, there were no effects on mean food consumption at any dose level and any study phase (pre-pairing, gestation, lactation)

REPRODUCTIVE PARAMETERS
Mating of all females was recorded during the first pairing period. The precoital time was not affected by the treatment, with a median precoital time of 3 days for each dose level tested.
All eleven paired females per group were mated. From the mated females, 11/11 (i.e. 100%) became pregnant in the control and the low dose group (100 mg/kg bw), whereas 10/11 (i.e., 90.9%) and 9/11 (i.e., 81.8%) became pregnant in the mid (300 mg/kg bw/day) and the high (1000 mg/kg bw/day) dose group, respectively. Thus, the fertility indices and conception rates showed no test item related impairments.
All pregnant females of the control, the mid and the high dose group, reared their pups until day 4 post partum. In the low dose group, 10/11 pregnant females reared the pups until day 4 post partum, since one female had lost her litter; the finding was not considered to be related to the treatment.
No effects on duration of gestation were observed at any dose level, since the mean duration of gestation was 21.4, 21.7, 21.6 and 21.8 days, at 0, 100, 300 and 1000 mg/kg bw/day, respectively.
No effects on corpora lutea count were observed at any dose level, since the mean number of corpora lutea per dam was 15.0, 14.6, 14.2 and 14.9, at 0, 100, 300 and 1000 mg/kg bw/day, respectively.
No effects on implantation rate and post-implantation loss were observed. In fact, the number of implantations per dam was 13.8, 13.3, 14.2 and 14.1, at 0, 100, 300 and 1000 mg/kg bw/day, respectively. The respective mean number of post-implantation loss per dam was 0.8, 1.1, 1.5 and 1.0, at 0, 100, 300 and 1000 mg/kg bw/day.

ORGAN WEIGHTS
No treatment-related changes in organ weights could be evidenced at any dose level tested.

GROSS PATHOLOGY
Neither the females nor the males showed macroscopic abnormalities related to the treatment. In fact, findings that occurred were seen in individual animals and/or were within the range of the normal background alterations.

HISTOPATHOLOGY
There were no microscopic findings that could be attributed to the treatment with the test item. In fact, all findings recorded were within the range of normal background alterations. All PAS-stained testes of the study were qualitatively staged. There were no abnormal lesions encountered during sperm staging regarding completeness of stages and maturation of cell populations. Individual lesions recorded were within the range of background alterations that may be recorded in this type of study, in rats of this strain and age.

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects on mating performance, fertility, corpora lutea count or duration of gestation were observed at any dose level.

Critical effects observed:

no

VIABILITY
There was no effect on mean value of living pups per dam at first litter check, thus, birth index was unaffected by treatment with the test item.
The overall mean numbers of living pups per dam at first litter check were 13.0, 12.2, 12.7 and 13.3, whereas birth indices (number of pups born alive as % of implantations) were 94.1%, 91.8%, 89.4% and 93.0% at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day, respectively.
There further was no effect on postnatal loss at any dose level. In fact, at 100 mg/kg bw/day, four pups were found dead in one litter at first check. The remaining pups (5 males and 5 females) in this litter were missing on day 2 post partum. This findings was considered to be incidental. Due to this total litter loss, the mean number of postnatal loss was statistically significantly higher at this dose level. The overall mean number of postnatal loss per dam was 0.2, 1.2, 0.5 and 0.3 at 0, 100, 300 and 1000 mg/kg bw/day, respectively.

EXTERNAL EXAMINATION
No abnormalities were noticed during the first litter check and during lactation at any dose level tested.

BODY WEIGHT
No effects on pup body weights were noted at any dose level. In fact, mean body weights of pups on day 1 post partum were 5.9, 6, 5.7 and 5.8 g, at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day respectively. The respective body weight gains of the pups during the first 4 days post parturition were +38.1%, +38.2%, +40.7% and +39.3%.

SEX RATIO
Pups sex ratio was not affected by the treatment at any dose level. At first litter check, percentages of male pups were 52%, 43%, 54% and 48%, at 0, 100, 300 and 1000 mg/kg bw/day, respectively.

PATHOLOGY
No abnormalities were noticed in the pups at any dose level tested.

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Generation:

F1

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: examination of litter data and of pups (weight, sex ratio, gross pathology) revealed no treatment-related changes indicative of developmental toxicity.

Critical effects observed:

no

Reproductive effects observed:

no

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:

valid data obtained from a screening study, sufficient for assessment of the reproduction toxicity potential of the test item.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In order to get preliminary information on possible effects of the test item Pigment Orange 79 on reproduction and development, a screening test according to the OECD TG 421 (1995) under GLP conditions was performed (BASF 80R0592/11X454, 2012). For this purpose, 4 groups of 11 male and 11 female Wistar rats were treated by gavage with the test item at following dose levels: 0, 100, 300 and 1000 mg/kg bw/day. In fact, the males were treated over a 14-day pre-pairing period and during the pairing period up to one day prior necropsy; the females were treated throughout the pre-pairing, pairing, gestation and lactation period up to the day 3 post partum. The control animals were dosed with the vehicle alone (purified water).

All animals survived the treatment, and stained/discolored red feces were the only clinical sign observed in the treated groups, which was due to the color of the test item and thus not a treatment-related adverse effect.

No treatment-related effects on food consumption, body weight and body weight gain were observed.

No treatment-related effects on reproductive parameters (e.g., mating performance, fertility, corpora lutea count, duration of gestation) were noticed.

No treatment-related effects on the pups were noticed.

Thus, based on the results of the present study, the NOAEL regarding systemic toxicity and reproductive toxicity in male and female Wistar rats was set at 1000 mg/kg bw/day, which was the highest dose level tested.

Short description of key information:
The test item Pigment Orange 79 was examined in the screening test according to the OECD TG 421 (1995) under GLP conditions (Harlan d51520, 2012). The NOAEL regarding systemic toxicity and reproductive toxicity in male and female Wistar rats was set at 1000 mg/kg bw/day, which was the highest dose level tested.

Justification for selection of Effect on fertility via oral route:
New guideline study conducted under GLP conditions

Effects on developmental toxicity

Description of key information

The test item Pigment Orange 79 was examined in the screening test according to the OECD TG 421 (1995) under GLP conditions (Harlan d51520, 2012). The NOAEL regarding developmental toxicity for Wistar rats offspring was set at 1000 mg/kg bw/day, which was the highest dose level tested.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

In the screening OECD TG 421 (1995) test conducted under GLP conditions and reported above (Harlan D51520, 2012), the offspring was examined for detection of any treatment-related changes or effects resulting from the treatment of the parental males and females with the test item Pigment Orange 79 by gavage, at dose levels of 0, 100, 300 and 1000 mg/kg bw/day. In fact, the parental males were treated over a 14-day pre-pairing period and during the pairing period up to one day prior necropsy, whereas the females were treated throughout the pre-pairing, pairing, gestation and lactation period up to the day 3 post partum. The control animals were dosed with the vehicle alone (purified water). Within the OECD 421 screening test, the litters were examined for litter size, live births, still births and any gross anomalies. The sex ratio of the pups was recorded, and the pups were weighed individually on day of parturition and on day 1 and 4 post partum. All pups were examined for any structural changes, either at the scheduled necropsy or during the study (day 1 to 4 post partum) if death occurred. Birth index (number of pups born alive as % of implantation) was determined.

The overall mean numbers of living pups per dam at first litter check were 13.0, 12.2, 12.7 and 13.3, whereas birth indices (number of pups born alive as % of implantations) were 94.1%, 91.8%, 89.4% and 93.0% at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day, respectively. There further was no effect on postnatal loss at any dose level. In fact, at 100 mg/kg bw/day, four pups were found dead in one litter at first check. The remaining pups (5 males and 5 females) in this litter were missing on day 2 post partum. This findings was considered to be incidental. Due to this total litter loss, the mean number of postnatal loss was statistically significantly higher at this dose level. The overall mean number of postnatal loss per dam was 0.2, 1.2, 0.5 and 0.3 at 0, 100, 300 and 1000 mg/kg bw/day, respectively. The examination of the pups revealed no treatment-related abnormalities. No effects on pup body weights were noted at any dose level. In fact, mean body weights of pups on day 1 post partum were 5.9, 6, 5.7 and 5.8 g, at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day respectively. The respective body weight gains of the pups during the first 4 days post parturition were +38.1%, +38.2%, +40.7% and +39.3%. The sex ratio was not affected by the treatment at any dose level, since at first litter check, percentages of male pups were 52%, 43%, 54% and 48%, at 0, 100, 300 and 1000 mg/kg bw/day, respectively. Necropsy revealed no abnormalities in the pups at any dose level tested.

Thus, regarding developmental toxicity, the NOAEL under the test conditions of the OECD TG 421 study can be set at 1000 mg/kg bw/day.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available OECD 421 screening test is reliable and suitable for the purpose of classification under Regulation (EC) No. 1272/2008. Based on the data, classification for reproduction and developmental toxicity is not warranted under Regulation (EC) no. 1272/2008.