A mixture of: N-(4-chlorophenyl)-4-(2,5-dichloro-4-(dimethylsulfamoyl)phenylazo)-3-hydroxy-2-naphthalenecarboxamide; N-(4-chlorophenyl)-4-(2,5-dichloro-4-(methylsulfamoyl)phenylazo)-3-hydroxy-2-naphthalenecarboxamide

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

- Rationale for reliability of the study report related to the source substance: Guideline study (OECD 421), GLP compliant (original reliability: 1) - Read across hypothesis: The similar chemical structure and the uniformity of physicochemical, environmental fate and toxicological properties justifies the application of read across to predict the outcome of a Developmental/Reproduction Screening Test and an In Vitro Mammalian Cell Gene Mutation Test (HPRT) of the target pigment based on available date coming from several source pigments.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for Testing of Chemicals, Section 4, No. 421, “Reproduction/Developmental Toxicity Screening Test”adopted on 27 July 1995.

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Health Effects guidelines, OPPTS 870.3550, Reproduction/ Developmental Toxicity Screening Test. EPA 712-C-00-367, July 2000.

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No. 440/2008, L 142, Annex Part B, May 30, 2008.

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

Test System:
Species/strain: Crl: WI(Han): Wistar rats (Full-Barrier)
Source: e.g. Charles River, 97633 Sulzfeld, Germany
Sex: Males and non-pregnant nulliparous females
Age at the beginning of the study: 10-11 weeks old
Body weight at the beginning of the study: interval within +/- 20% of the mean weight.
The range of the body weight was:
Females: 174-220 g, (mean: 199.10 +/- 20%= 39.82 g)
Males: 270-321 g, (mean: 292.90 g, +/- 20%= 58.58 g)
Number of animals: 10 Males and 10 females per group
The animals were derived from a controlled full barrier maintained breeding system (SPF). According to Art. 9.2, No.7 of the German Act on Animal Welfare the animals are bred for experimental purposes.

Housing and Feeding Conditions:
After an adequate acclimatisation period (at least five days) the animals were barrier maintained (full-barrier) in air conditioned rooms under the following conditions:
- Temperature: 22 +/- 3 °C
- Relative humidity: 55 +/- 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice (Lot No.1151)
- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiol. controlled periodically)
-housed individually in IVC cages (except during the mating period when one female was paired with one male), type III H, polysulphone cages on Altromin saw fibre bedding (Lot No.300512)
Certificates of food, water and bedding are filed at BSL BIOSERVICE.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

The animals were dosed with the test item on 7 days per week for a period of approximately 54 days. The test item was administered daily during 14 days pre mating and 14 days mating period in both male and in female, during gestation period and up to post natal day 3 in females. Males were dosed for 28-31 days.

The test item was administered by gavage using a gavaging canula. The maximum dose volume administered was 5 mL / kg body weight.

For each animal, the individual dosing volume was calculated on the basis of the most recently measured body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples for the nominal concentration verification were taken in study week 1 (first week of pre mating period), study week 3 (first week of mating), study week 5 (gestation) and study week 7 (gestation/lactation) - (total 16 samples)

Samples for homogeneity were taken from the top, middle and bottom of the high dose, medium dose and low dose preparation in study week 1 and 5 (total 18 samples)

All samples were stored at -20°C until analysis. Sample quantity for all samples was 10 mL in a 15mL falcon tube.

All samples of dosing formulations were sent to Analytic department of BSL BIOSERVICE Scientific Laboratories GmbH after completion of in-life phase.

Details on mating procedure:

Animal were paired in the ratio of 1:1 (male to female). The subsequent morning and the next morning there onwards the vaginal smear of the females were checked to confirm the evidence of mating. Day of vaginal plug and/or sperm was considered as day 0 of gestation.

Duration of treatment / exposure:

Males 28-31 days; Females: Approx. 54 days

Frequency of treatment:

7 days/week

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/day
Basis:
other: content C.I.

No. of animals per sex per dose:

10 animals/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

Number and Sex of the Animals:
80 animals (40 males and 40 females) were included in the study (10 male and 10 female animals per group). The study included three dose groups (LD, MD and HD) and one control group (C).

Preparation of the Animals:
Prior to the start of the treatment period a detailed clinical observation outside the home cage was made. Animals were healthy and none of them shown any pathological signs before the first administration. Before the first administration, all animals to be used for the study were weighed and assigned to the experimental groups with achieving a most homogenous variation in body weight throughout the groups of males and females. The animals were acclimatised for at least five days before the first dose administration.

Experimental group and Dosage:
In consultation with the sponsor and based on the a BSL dose range finding study, doses levels of 100, 300, 1000 were selected for the 3 dose groups (LD, MD and HD) and 1 control group (C)
Dose concentration was based on the purity/content of the test item (98.2 %)
The animals in the control group were handled in an identical manner to the dose group subjects and received corn oil in the same volume as used for treatment groups.

Administration of Doses:
The animals were dosed with the test item on 7 days per week for a period of approximately 54 days. The test item was administered daily during 14 days pre mating and 14 days mating period in both male and in female, during gestation period and up to post natal day 3 in females. Males were dosed for 28-31 days.
The test item was administered by gavage using a gavaging canula. The maximum dose volume administered was 5 mL / kg body weight.
For each animal, the individual dosing volume was calculated on the basis of the most recently measured body weight.

Mating:
Animal were paired in the ratio of 1:1 (male to female). The subsequent morning and the next morning there onwards the vaginal smear of the females were checked to confirm the evidence of mating. Day of vaginal plug and/or sperm was considered as day 0 of gestation. Cages were arranged in such a way that possible effects due to cage placement was minimised.

Clinical Observation:
Animals were observed for clinical signs during the entire treatment period. General clinical observations were made at least once a day, preferably at the same time each day and considering the peak period of anticipated effects after dosing. The health condition of the animals was recorded. Twice daily (and once daily during weekends and holidays) all animals were observed for morbidity and mortality. pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity were recorded.

Body Weight and Food Consumption:
The body weight of all animals was recorded once before assignment to the experimental groups and on the first day of administration.
In the male animals, the body weight was taken weekly during the entire study period and on day of terminal sacrifice.
In the female animals the body weight were taken weekly during the pre-mating period, on gestation day 0, 7, 14, 20 and on post-natal day 0 (within 24 hours of parturition) and post-natal day 4 along with pups.

Food consumption was measured on the corresponding day of the body weight measurements after the beginning of the dose administration. Food consumption was not measured during the mating period and post mating period in males.

Litter observations:
The duration of gestation was recorded and is calculated from day 0 of pregnancy. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities.

Live pups were counted and sexed and litters weighed within 24 hours of parturition (day 0 post-partum) and on day 4 post-partum. Live pups were identified by writing actual numbers on the back with the help of permanent marker In addition to the observations on parent animals, any abnormal behaviour of the offspring was recorded.

Sperm Analysis:
At necropsy (one day after the last administration) one epididymis and one testis was separated and used for evaluation of sperm parameters. Epididymal sperm motility and testicular sperm count was evaluated in all male animals using Hamilton Thorn Sperm Analyser (TOX IVOS Version 13.0 C).

Gross Pathology:
Males were sacrificed after the completion of mating period (total dosing of 28-31 days) and females were sacrificed on respective post natal day 4 along with pups At the time of sacrifice or death during the study, the adult animals were examined macroscopically for any abnormalities or pathological changes. Special attention was paid to the organs of the reproductive system.
Pups sacrificed on day 4 post-partum were carefully examined for gross external abnormalities.
The number of implantation sites and corpora lutea was recorded in all pregnant females by gross observations.
The ovaries, uterus with cervix, vagina, testes, epididymides, accessory sex organs (prostate, seminal vesicles with coagulating glands as a whole) were preserved in 10 % neutral buffered formalin. Testes and epididymides were initially preserved in modified Davidson’s Solution for approximately 24 hours and transferred to 10 % neutral buffered formalin.

Organ Weight:
The testes, epididymides, prostate and seminal vesicles with coagulating gland of all male adult animals and ovaries, uterus with cervix of all female adult animals were weighed.
Paired organs were weighed separately.

Histopathology:
A full histopathological evaluation (after the preparation of paraffin sections and haematoxylin-eosin staining) was carried out on all animals of the control and high dose groups which are sacrificed at the end of the treatment period.
Histopathological examinations were not extended to animals of the other dose groups, as no treatment-related changes were observed in the high dose group.
A detailed qualitative examination of the testes was made taking into account the tubular stages of the spermatogenic cycle at the evaluation of additional haematoxylin-PAS (Periodic Acid Schiff) stained slides.
Gross lesions macroscopically identified were examined microscopically.
The histological processing of tissues to microscope slides were performed at the GLP-certified contract laboratory Propath UK Ltd, Willow Court, Netherwood Road, Hereford HR2 6JU, Great Britain (test site for tissue processing). The histopathological evaluation was performed at the GLP-certified contract laboratory KALEIDIS- Consultancy in Histopathology (test site for histopathology), 6 rue du Gers, 68300 Saint-Louis, France. Blocking, embedding, cutting, H&E staining and scientific slide evaluation was performed according to the corresponding SOP’s of the test sites.

Examinations

Maternal examinations:

Body weight, food consumption, clinical signs,macropscopic findings, micropscopic findings, reproductive behaviour (copulation index, fertility index)

Ovaries and uterine content:

macropscopic and micropscopic examination.

Fetal examinations:

Number, weight and sex of pups, No. of still births, live births, runts and gross external abnormalities on PND 0 and 4.

Statistics:

A statistical assessment of the results of the body weight, food consumption, litter data and absolute and relative organ weights was performed for each gender by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. These statistics were performed with GraphPad Prism V.5.01 software (p<0.05 is considered as statistically significant).

Indices:

Copulation, Fertility, Delivery and Viability Indices

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
Mortality:
No mortalities were observed at any dose levels of male and female animals.

Clinical Observation:
No test item related clinical signs were observed in male and female animals. Few spontaneous clinical signs observed occasionally in male and female animals were alopecia at forepaws (1/10 in Control males and 1/10 in LD, MD and HD females), red nasal discharge (2/10 in HD males), slight piloerection (1/10 in LD, HD males, 1/10 in LD females). Discoloured red faeces were observed in all male and female treatment group animals throughout the study. This discoluration of the faeces was attributed to the red colour of the test item and as such of no toxicological significance.

Body Weight and Body Weight Change:
In male, no statistically significant effect on body weight and body weight change was observed throughout the study period in treatment groups when compared with controls.
In females, statistical analysis of body weight and body weight change data revealed no significant difference in treatment groups during premating, gestation and lactation period when compared with controls.

Food Consumption:
In males and females, statistical analysis of food consumption data revealed no test item related effect on food consumption in treatment groups during entire study period when compared with controls.
In correlation to the body weight and body weight change, the food consumption in both males and females increased with the progress of the study in all groups.

Gross Pathology:
At necropsy of male (after minimum total dosing of 28 days - on day 29) and females (on post-natal day 4) by using a high dose of Ketamine/Xylazine (2:1), macroscopic examination of the animals revealed no test item related macroscopic findings in males and females. Few spontaneous gross pathological findings observed in male and female animals were yellow spot on right epididymides (1/10 in MD males), cyst on left ovary (1/10 in LD females), cyst on right ovary (1/10 in LD and MD females), cyst on both ovaries (1/10 in MD females), white areas on lung (1/10 in MD females) and extra red tissue on thymus (1/10 in MD females).
These gross pathological findings were spontaneous in nature and as such not a systemic effect due to the test item administration.

Organ Weight:
In males and females, no statistically significant effect on absolute and relative (to body weight) organ weights was observed in any treatment group when compared with the controls.

Histopathology
At terminal sacrifice, macroscopic organ findings were very few and none of them was considered to indicate a test item-related toxic effect.
No test item-related histological findings were noted in the male and female reproductive organs. Female reproductive organs showed similar post-partum histomorphology in the control and high dose group. The number of large ovarian corpora lutea was not essentially different between control animals and animals treated at 1000 mg/kg/day.
Two control females, one female treated at 100 mg/kg/day and one female treated at 1000 mg/kg/day were found not to be pregnant at terminal sacrifice, but this was not considered to be test item-related.
As a conclusion, under the conditions of the present study and based on the histopathological evaluation as defined by the study plan, the NOAEL (No Observed Adverse Effect Level) for pathology is considered to be 1000 mg/kg/day.

Dose Formulation Analysis:
Concentration analysis of formulation samples was determined in study week 1, 4, 5, and 7 for all dose groups. The mean recoveries observed in LD, MD and HD groups were 88.1%, 91.2%, and 86.6% of the nominal concentration, respectively.
Homogeneity of formulation samples was determined in study week 1 and 5 for all dose groups. The mean recoveries observed for LD group were 93.3% and 102.3%, for MD group 98.2% and 103.2%, and for HD group 99.1% and 100.3% of the nominal value.

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

other: content C.I. (nominal)

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

other: content C.I. (nominal)

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Soft tissue and skeletal examinations were not made.

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Litter Weight Data: Group mean litter weight, total litter weight, male and female litter weight on PND 0 and 4 remained unaffected in all treated groups when compared with controls. Precoital Interval and Duration of Gestation: No treatment related effect was observed on precoital interval and duration of gestation and values were comparable between the groups. All pregnancies resulted in normal births. Successful mating resulted in 8, 9, 10 and 9 pregnancies in the control, low, mid and high dose respectively Pre and Post Natal Data: Pre and post natal data like group mean number of corpora lutea, number of implantation sites, number of pups born on PND 0, percent preimplantation and post implantation loss remained unaffected due to treatment when compared with controls. Litter Data: No treatment related effect was observed on total number of pups born, number of males, number of females, sex ratio, live pups, still birth and runt on PND 0 and number of males, number of females, total number of pups and sex ratio on PND 4. All group mean and individual values for various litter data parameters from treatment groups were comparable with the controls. Reproductive Indices: No treatment related effect on copulation index, delivery index, fertility index and viability index was observed when compared with controls. Reduced fertility index (No. of pregnant females/No. of copulated females X 100) was observed in C (80%), LD and HD (90 %) dose group as compared MD group (100 %). Pup Survival Data: Survival of the pups from PND 0 to PND 4 remained unaffected due to the treatment in all treatment groups except one pup mortality from HD female 72 (Pup No. 3) was observed on PND 1. Two pups, one each from LD and MD group female 58 (pup No. 3) and female 67 (pup No. 3) went missing on PND 3 and those were presumed to be cannibalized by the dam. Since this incidence of cannibalism was observed in one female of the two intermediate groups and it was within the rat cannibalism rate, this incidence was not considered to be test item related. Pup External Finding: No treatment related gross external findings were observed in pups from any of the treated groups on PND 0 and 4. However, few spontaneous findings were observed and were not related to the treatment with test item.

Read across justification

The purpose of this assessment is to provide justification for read across in order to predict the potential genetic and reproductive toxicity of the target substance Pigment Orange 74 based on available date coming from a set of source substances (Pigment Red 22, 112, 146, 147, 179).

The pigments used for read across are structurally similar and differ only by different substituents in the common core molecule. Target and source substances are solids which decompose or melt at high temperatures (>= 237°C). Solubility in water or n-octanol is very low or low (< 12 μg/L and < 3.4 mg/L, respectively). For the target substance solubility in water was determined to be below the limit of detection (<0.1 mg/L) and in n-octanol 0.16 mg/L. These values suggest poor absorption and low bioavailability for all pigments involved in this evaluation. The log n-octanol-water partition coefficients are in the range of 1.28 to 2.5 for all of the source pigments which fit together with log n-octanol-water partition coefficient of Pigment Orange 74 (>0.2). These values are far below the limit of concern considered to be critical for bio-accumulative properties. All pigments showed very limited biodegradability, which is assumed to be due to their unavailability for microorganisms. Lacking bioavailability is probably also the reason for the absence of any relevant mammalian toxicity: None of the source pigments showed any toxic effects after single oral or dermal or after repeated oral exposure including effects on reproductive performance (examined for Pigment Red 22 and 170). This pertains also for the target substance with the exception of one death in association with lung coloration observed after single oral application (2000 mg/kg bw) which might be attributed to incorrect dosing or inhaling of foamed vomit. A spontaneous not treatment related death cannot be ruled out as well. This seems not unlikely in view of the fact that no such effects were observed in an oral repeated dose study. Target and source pigments have no skin sensitising effects. All pigments are not mutagenic in the Bacterial Reverse Mutation Assay or the Mammalian Chromosomal Aberration Test. Negative results were also obtained for the source substances in the In vitro Mammalian Cell Gene Mutation Test (HPRT).

Due to the very low solubility of the pigments it can reasonably be assumed that the members of this read across approach are (nearly) not present in a dissolved form on the skin or mucous membranes after dermal, oral or inhalative exposure, i.e. they could not be absorbed via skin and mucous membranes. This conclusion is supported by the observation that the pigments evaluated do not exert any relevant toxicity.

This assessment is based on experimental data on the source pigments as compared to available data of the target pigment covering the following endpoints: Acute oral or dermal toxicity, skin and eye irritation, skin sensitisation, genotoxicity in vitro, subacute oral toxicity, toxicity to reproduction, and inherent biodegradability (for details see data matrix).

In conclusion, the uniformity of physicochemical, environmental fate and toxicological properties justifies the application of read across to predict the outcome of a Developmental/Reproduction Screening Test and an In Vitro Mammalian Cell Gene Mutation Test (HPRT) of the target pigment based on available date coming from several source pigments. The minor differences in the structure do not significantly alter the basic physicochemical properties or the basic biological effects.

DataMatrix

CHEMICAL NAME	Pigment Orange 74	Pigment Red 22	Pigment Red 112	Pigment Red 146	Pigment Red 147	Pigment Red 170
Role	Target Substance	Source Substance	Source Substance	Source Substance	Source Substance	Source Substance
CAS No.	85776-14-3	6448-95-9	6535-46-2	5280-68-2	68227-78-1	2786-76-7
PHYSICAL AND CHEMICAL PROPERTIES
State of the substance at 20° C and 101,3 kPa	orange solid	red solid	red solid	red solid	red solid	red solid
Melting/freezing point	>= 278.2°C, <= 304.9°C	decomp. starting at 237°C, 534 J/g	decomp. starting at 270°C, 300 J/g	decomp. starting at 283°C, 418 J/g	decomp. starting at 278°C, 92 J/g	decomp. starting at 313°C, 80 J/g
Water solubility	< 0.1 mg/L (below the detection limit)	11.8 μg/L	9.80 μg/L	8.7 μg/L	10 μg/L	11.9 μg/L
n-Octanol solubility	0.16 mg/L   0.192 mg/100 g fat	1.80 mg/L	3.31 mg/L	0.100 mg/L	0.74 mg/l	0.225 mg/L
log Partition coefficientn-octanol/water	> 0.2 (exact value could not be determined because water solubility was below the detection limit)	2.18	2.5	1.87	1.87	1.28
Stability in organic solvents and identity of relevant degradation products	>72 h in DMSO and 1,2-propylene glycol	>72h in DMSO and 1,2-propylene glycol	>72h in DMSO and sesame oil	>72h in DMSO and 1,2-propylene glycol	>72h in DMSO and 1,2-propylene glycol	>72h in DMSO and 1,2-propylene glycol
TOXICOLOGICAL INFORMATION
Skin irritation	not irritating	not irritating (in vitro)	not irritating	not irritating	not irritating (read across)	not irritating
Eye irritation	not irritating	not irritating (read across) b	not irritating	not irritating	not irritating (read across)	not irritating
Skin sensitization	not skin sensitising	not skin sensitising (read across)	not skin sensitising	not skin sensitising	not skin sensitising	not skin sensitising
In vitrogene mutation study in bacteria	not mutagenic	not mutagenic	not mutagenic	not mutagenic	not mutagenic	not mutagenic
In vitrocytogenicity study in mammalian cells	not mutagenic (CA in V79 cells)	not mutagenic (CA in V79 cells)	not mutagenic (MN in V79 cells)	not mutagenic (CA in V79 cells)	not mutagenic (CA in V79 cells)	not mutagenic (CA in V79 cells)
In vitrogene mutation study in mammalian cells	RA-conclusion: not mutagenic (HPRT in V79 cells)	not mutagenic (read across)	Applied Source Substance: not mutagenic (HPRT in V79 cells)	Applied Source Substance: not mutagenic (HPRT in V79 cells)	Applied Source Substance: not mutagenic (HPRT in V79 cells)	Applied Source Substance: not mutagenic (HPRT in V79 cells)
Otherin vivomutagenicity tests	No data requirement	not mutagenic (read across)	not mutagenic (read across)	not mutagenic (read across)	not mutagenic (UDS in vivo)	not mutagenic (read across)
Acute toxicity, oral route, (LD50 mg/kg b.w., rats)	> 2000	> 2000 (read across) a	> 5000 (male/female)	> 10000 (female)	> 2000 (read across)	> 15000 (male/female)
Acute tocity, inhalation (LC50 mg/L, rats )	Waiving (according to REACH Annex VIII, no.8.5, column 2; data for 2 application routes of acute toxicity available)	Waiving (according to REACH Annex VIII, no.8.5, column 2; data for 2 application routes of acute toxicity available)	Waiving (according to REACH Annex VIII, no.8.5, column 2; data for 2 application routes of acute toxicity available)	Waiving (according to REACH Annex VIII, no.8.5, column 2; data for 2 application routes of acute toxicity available)	Waiving (according to REACH Annex VIII, no.8.5, column 2; data for 2 application routes of acute toxicity available)	Waiving (according to REACH Annex VIII, no.8.5, column 2; data for 2 application routes of acute toxicity available) (>1580 mg/m3/4h; RL3)
Acute toxicity dermal route (LD50 mg/kg b.w., rat)	> 2000 (male,female)	> 2000 (read across)	> 5000 (male)	> 2000 (read across)	> 2000  (read across)	> 2000 (male,female)
Short-term repeated dose toxicity study in rats (oral)	NOAEL 1000 mg/kg bw (highest dose tested; OECD 407)	NOAEL 1000 mg/kg bw (highest dose tested; OECD 422)	NOAEL 1000 mg/kg bw (highest dose tested; OECD 407)	NOAEL 1000 mg/kg bw (highest dose tested; OECD 407)	NOAEL 1000 mg/kg/day (read across)	NOAEL ca. 1200 mg/kg bw (highest dose tested; OECD 407)
Sub-chronic toxicity study in rats (oral)	waiving (Annex XI: scientifically unjustified, because substances are not bioavailable and show no toxic effects in acute and short-term repeated dose studiesàpredicted not to be toxic after long-term repeated exposure)	waiving (Annex XI: scientifically unjustified, because substances are not bioavailable and show no toxic effects in acute and short-term repeated dose studiesàpredicted not to be toxic after long-term repeated exposure)	waiving (Annex XI: scientifically unjustified, because substances are not bioavailable and show no toxic effects in acute and short-term repeated dose studiesàpredicted not to be toxic after long-term repeated exposure)	waiving (Annex XI: scientifically unjustified, because substances are not bioavailable and show no toxic effects in acute and short-term repeated dose studiesàpredicted not to be toxic after long-term repeated exposure)	Waiving (Annex XI: scientifically unjustified, because substances are not bioavailable and show no toxic effects in acute and short -term repeated dose studiesàpredicted not to be toxic after long-term repeated exposure)	Waiving (Annex XI: scientifically unjustified, because substances are not bioavailable and show no toxic effects in acute and short-term repeated dose studiesàpredicted not to be toxic after long-term repeated exposure)
Carcinogenicity	No data requirement	No data requirement	No data requirement	No data requirement	No data requirement	No data requirement
Screening for reproduction/developmental toxicity, rats	RA-conclusion: NOAEL 1000 mg/kg bw	Applied Source Substance: NOAEL 1000 mg/kg bw (highest dose tested; OECD 422)	NOAEL 1000 mg/kg/day (read across)	NOAEL 1000mg/kg/day (read across)	NOAEL 1000 mg/kg/day (read across)	Applied Source Substance: NOAEL 1000 mg/kg bw (highest dose tested; OECD 421)
Reproductive Toxicity – Fertility	Waiving (Annex XI: scientifically unjustified, because substances are not bioavailable and show no toxic effects in acute and repeated dose and reproduction / developmental screening studiesàpredicted not to be toxic to reproduction)	Waiving (Annex XI: scientifically unjustified, because substances are not bioavailable and show no toxic effects in acute and repeated dose and reproduction / developmental screening studiesàpredicted not to be toxic to reproduction)	Waiving (Annex XI: scientifically unjustified, because substances are not bioavailable and show no toxic effects in acute and repeated dose and reproduction / developmental screening studiesàpredicted not to be toxic to reproduction)	Waiving (Annex XI: scientifically unjustified, because substances are not bioavailable and show no toxic effects in acute and repeated dose and reproduction / developmental screening studiesàpredicted not to be toxic to reproduction)	Waiving (Annex XI: scientifically unjustified, because substances are not bioavailable and show no toxic effects in acute and repeated dose and reproduction / developmental screening studiesàpredicted not to be toxic to reproduction)	Waiving (Annex XI: scientifically unjustified, because substances are not bioavailable and show no toxic effects in acute and repeated dose and reproduction / developmental screening studiesàpredicted not to be toxic to reproduction)
Reproductive Toxicity – Developmental Toxicity	Waiving (Annex XI: scientifically unjustified, because substances are not bioavailable and show no toxic effects in acute and repeated dose and reproduction / developmental screening studiesàpredicted not to be developmental toxic)	Waiving (Annex XI: scientifically unjustified, because substances are not bioavailable and show no toxic effects in acute and repeated dose and reproduction / developmental screening studiesàpredicted not to be developmental toxic)	Waiving (Annex XI: scientifically unjustified, because substances are not bioavailable and show no toxic effects in acute and repeated dose and reproduction / developmental screening studiesàpredicted not to be developmental toxic)	Waiving (Annex XI: scientifically unjustified, because substances are not bioavailable and show no toxic effects in acute and repeated dose and reproduction / developmental screening studiesàpredicted not to be developmental toxic)	Waiving (Annex XI: scientifically unjustified, because substances are not bioavailable and show no toxic effects in acute and repeated dose and reproduction / developmental screening studiesàpredicted not to be developmental toxic)	Waiving (Annex XI: scientifically unjustified, because substances are not bioavailable and show no toxic effects in acute and repeated dose and reproduction / developmental screening studiesàpredicted not to be developmental toxic)
Toxicokinetic behaviour	Available data point to inert behavior and non-bioavailability	Available data point to inert behavior and non-bioavailability	Available data point to inert behavior and non-bioavailability	Available data point to inert behavior and non-bioavailability	Available data point to inert behavior and non-bioavailability	Available data point to inert behavior and non-bioavailability
ENVIRONMENTAL FATE
Inherent biodegradability	Not readily biodegradable	Not inherently biodegradable (read across)	Not inherently biodegradable (read across)	Not inherently biodegradable (read across)	Not inherently biodegradable (read across)	Not inherently biodegradable (read across)

Applicant's summary and conclusion

Conclusions:

In conclusion, the repeated dose administration of the test item to the male (28 days) and female (maximum 54 days) Wistar rats at dosages of 0, 100, 300 and 1000 mg/kg body weight revealed neither mortalities nor findings of toxicological relevance.
Based on the data generated from this reproduction/ developmental toxicity screening test, the no observed adverse effect level (NOAEL) is believed to be 1000 mg/kg body weight for reproduction/ developmental toxicity screening in males and females.

Based on these findings the test item is not subject to classification and labelling.

Executive summary:

The aim of this study was to assess the possible effect of the test item on male, female fertility and embryofetal development in Wistar rat. This study was also aimed to provide initial information on possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus and parturition.

In this study, four groups comprised of 10 adult males and 10 non pregnant nulliparous female rats (Wistar Crl:WI) were dosed daily by oral gavage with 100, 300 and 1000 mg/kg body weight per day of test item at dose volume of 5 mL/kg body weight. The test item was formulated in sterile water. Control animals were handled identically as treated groups and received sterile water in similar volume as treated groups.

Doses evaluated were: 0, 100, 300, 1000  mg/kg body weight/day

Dose concentration was based on the purity/content (content C.I.) of the test item (98.2 %).

The test item formulation was prepared freshly and administered daily during 14 days pre mating and 14 days mating period in both male and in female, during gestation period and up to post natal day 3 in females. Males were dosed for total period of 28 days. Dose volumes were adjusted weekly based on the recent body weight measurement.

Animals were examined daily for the clinical signs and mortality. Body weight and food consumption was measured weekly except during the mating period and post mating period in males where food consumption was not measured.

After 14 days of premating treatment to both male and female, animals were paired (1:1) for 14 days. The subsequent morning onwards, the vaginal smears of females were checked to confirm the evidence of mating in the form of sperm positive vaginal smears. After the confirmation of the mating, females were separated and housed individually. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities. Live pups were counted, sexed and litters were weighed within 24 hours of parturition and on day 4 post-partum.

Males and females were sacrificed on treatment day 29 and post natal day 4 respectively and subjected to necropsy. Non pregnant females (45, 49, 57 and 74) were sacrificed on respective day 26 after the evidence of mating. The wet weight of male and female reproductive organs was taken and preserved in 10 % neutral buffered formalin except testes and epididymides which were initially fixed in Modified Davidson’s fixative for approximately 24 hours before they were transferred to 10% neutral buffered formalin. Histopathological evaluation of the tissues was performed on high dose and control animals. Organs showing gross alterations were also examined histopathologically.

 

Summary Results

Clinical Signs and Mortality: No test item related clinical signs and mortalities were observed in both males and females.

Body Weight Development: In male, no statistically significant effect on body weight and body weight change was observed throughout the study period in treatment groups when compared with controls. In females, statistical analysis of body weight and body weight change data revealed no significant difference in treatment groups during premating, gestation and lactation period when compared with controls.

Food Consumption: In males and females, statistical analysis of food consumption data revealed no test item related effect on food consumption in treatment groups during entire study period when compared with controls.

Litter Weight Data: Group mean litter weight, total litter weight, male and female litter weight on PND 0 and 4 remained unaffected in all treated groups when compared with controls.

Precoital Interval and Duration of Gestation: No treatment related effect was observed on precoital interval and duration of gestation and valueswere comparable between the groups. All pregnancies resulted in normal births.

Pre and Post Natal Data: Pre and post natal data like group mean number of corpora lutea, number of implantation sites, number of pups born on PND 0, percent preimplantation and post implantation loss remained unaffected due to treatment when compared with controls.

Litter Data: No treatment related effect was observed on total number of pups born, number of males, number of females, sex ratio, live pups, still birth and runt on PND 0 and number of males, number of females, total number of pups and sex ratio on PND 4. All group mean and individual values for various litter data parameters from treatment groups were comparable with the controls.

Reproductive Indices: No treatment related effect on copulation index, delivery index, fertility index and viability index was observed when compared with controls.

Pup Survival Data: Survival of the pups from PND 0 to PND 4 remained unaffected due to the treatment in all treatment groups except one pup mortality from HD female 72 (Pup No. 3) was observed on PND 1. Two pups, one each from LD and MD group female 58 (pup No. 3) and female 67 (pup No. 3) went missing on PND 3 and those were presumed to be cannibalized by the dam.

Pup External Findings: No treatment related gross external findings were observed in pups from any of the treated groups on PND 0 and 4. However, few spontaneous findings were observed which were not considered to be test item related.

Sperm Analysis: Statistical analysis of sperm motility and testicular sperm head count data revealed no test item related effect on sperm parameters and all group mean and individual values from treatment groups were comparable with the controls.

Gross Pathology: At necropsy, macroscopic examination of the animals revealed no test item related macroscopic findings in males and females. Fewspontaneous gross pathological findings were observed in male and female animals and as such not a systemic effect due to the test item administration.

Organ Weight:In males and females, no statistically significant effect on absolute and relative (to body weight) organ weights was observed in any treatment group when compared with the controls.

Histopathology: At terminal sacrifice, macroscopic organ findings were very few and none of them was considered to indicate a test item-related toxic effect. No test item-related histological findings were noted in the male and female reproductive organs.

Dose Formulation Analysis: Concentration analysis of formulation samples was determined in study week 1, 4, 5, and 7 for all dose groups. The mean recoveries observed in LD, MD and HD groups were 88.1%, 91.2%, and 86.6% of the nominal concentration, respectively. Homogeneity of formulation samples was determined in study week 1 and 5 for all dose groups. The mean recoveries observed for LD group were 93.3% and 102.3%, for MD group 98.2% and 103.2%, and for HD group 99.1% and 100.3% of the nominal value.

 

The NOAEL for developmental/parental toxicity is1000 mg/kg bw/d.