Barium bis[2-chloro-5-[(2-hydroxy-1-naphthyl)azo]toluene-4-sulphonate]

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

The test material, D&C Red No. 9, was administered to rats in the diet at concentrations of 100, 200 and 500 ppm (equ. to 8, 14 and 42 mg/kg bw/day) for 8 weeks prior to mating (CFTA 1982). The treatment was continued during gestation and lactation. Rats from parents treated with graded dietary concentrations of D&C Red No. 9 (0, 100, 200 and 500 ppm) have been continued on treatment for 30 months after weaning. The test material was judged not to have an effect on body weight, food consumption, or fertility of the F0 generation rats. Similarly, no effect was evident on the viability or growth of F1 pups from birth to weaning. In a multi-generation reproduction study the test item was fed in the diet at dosage levels of 0. 05, 0.5, 1. 5 and 5.0 mg/kg/day to male and female rats. No changes seen for parental rats or pups with respect to general behavior or appearance, body weight or survival. The fertility, gestation, viability and lactation indices of all litters were comparable to control. Abnormal gross anatomical changes or microscopic pathologic lesions were not observed.

Link to relevant study records

Reference

Endpoint:

one-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August 1977

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Principles of method if other than guideline:

the study was not conducted as a one-generation study but meets basic principles of this test (exposure duration F0 animals, dose level, observations); histopathology of parental target organs and analysis of oestrous-cycle/spermatogenesis was not performed

GLP compliance:

no

Species:

rat

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Portage, Michigan
- Age at study initiation: (P) x 40d
- Housing: singly until mating (8 weeks), 1:1 for mating 7d, again separated for pregnancy, delivery and lactation (21d), F1 in groups
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 18d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23+/- 1
- Photoperiod (hrs dark / hrs light): 12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): The appropriate quantity (by weight) of D&C Red No. 9 (corrected for purity) was manually mixed with 5 kg of basal diet. This premix was mixed with approximately one-half the final volume of basal feed for 15 minutes in a twin shell blender. The remaining basal feed was then added, and mixing was continued for 15 minutes. From Weeks 1-15, 36 kg of diet were prepared at each dose level. From Week 15 until weaning of the pups, the diet was prepared in 54 kg batches as frequently as needed to supply adequate feed for the rats.
- Storage temperature of food: room temperature

Details on mating procedure:

- M/F ratio per cage: one to one
- Length of cohabitation: 7d
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): singly for pregnancy, delivery and lactation

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The homogeneity of the preparations was determined by analyzing 9 discrete samples from each dietary level of the first batch. Additionally, samples of the first batch were analyzed after storage at room temperature (25°C) for 7, 14 and 21 days and 7 days at 370C. During the remainder of the in utero phase of the study, the test diet was analyzed approximately weekly. The stability of D&C Red No. 9 in the diet under animal room conditions was determined by the analysis of diet remaining in 3 feed jars from each dietary level at the end of Weeks 2, 5, and 12. At Week 10 only, samples of the 100 ppm diet were analyzed to check for low values found in Weeks 2 and 5.

Duration of treatment / exposure:

8 weeks before mating and during mating (P), 30 month (whole life time) of the F1 generation

Frequency of treatment:

daily

Details on study schedule:

- Selection of parents from F1 generation when pups were 21 days of age.
- Age at mating of the mated animals in the study: 9.5 weeks

Dose / conc.:

100 ppm (nominal)

Dose / conc.:

200 ppm (nominal)

Dose / conc.:

500 ppm (nominal)

No. of animals per sex per dose:

60

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: no data
- Rationale for animal assignment: random

Positive control:

no

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations: physical appearance, signs of toxicity and mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
in weekly intervals until mating was initiated

days of gestation

Oestrous cyclicity (parental animals):

no data

Sperm parameters (parental animals):

no data

Litter observations:

bodyweight: The pups were sexed and weighed as a litter and the means were calculated on Days 0, 4 and 14. On Day 21, they were sexed and individual body weights were recorded. Body weights and food consumption were determined weekly for Weeks 1-14, subsequently at biweekly intervals over one week for Weeks 16-26, and at monthly intervals thereafter. Body weights were taken for all animals prior to necropsy

food consumption: Food consumption for dam and litter was determined for Days 0-4, 5-14, and 15-21.

clinical observation: Detailed observations were performed on all pups on Days 0, 4, 14 and 21 of lactation. After weaning and throughout the chronic phase, the animals were observed at least twice daily for general physical appearance, signs of toxicity and mortality.

ophthalmoscopic observation: The eyes of all surviving rats were examined at the initiation of the chronic phase, and at Months 3, 6, 12, 18 and 2

pathologic evaluation: Clinical pathologic evaluation was performed at Months 3, 6, 12, 18 and 24 of the chronic phase of the study. Rats scheduled for clinical pathology were fasted overnight prior to collection of the samples.

Hematology: hb, hc, RBC, white blood cell count, reticulocyte count. Specimens were collected by orbital sinus bleeding for blood chemistry and hematology samples

Clinical chemistry: glucose, BUN, SGOT, SGPT, alkaline phophatase, creatinine, total protein

urinalysis: color, appearance, specific gravity, protein, pH, ketones, bilirubin, glucose, occult blood and microscopic sediment was performed at the same intervals as the clinical pathologic testing. Urine was collected in stainless steel collection cages.

STANDARDISATION OF LITTERS
One male and i female pup were randomly selected from each litter when possible. To provide 70 animals per sex per dose group, litters were selected randomly to contribute an additional male and/or female pup which was selected randomly from the remaining pups

GROSS EXAMINATION OF DEAD PUPS:
no

PARAMETERS EXAMINED
The following parameters were examined: live pubs, dead pubs, sex, mean pub weight, on days 0, 4, 14 and 21

Postmortem examinations (parental animals):

SACRIFICE
Pups not selected for the chronic study, and the F0 generation rats were discarded

GROSS NECROPSY
- no

HISTOPATHOLOGY / ORGAN WEIGHTS
no

Postmortem examinations (offspring):

SACRIFICE
All animals dying on study or killed (via carbon dioxide) were necropsied. For the 12 month interim kill, 10 animals/sex/dose group were randomly
selected from the survivors and all surviving animals were killed at the 30 month terminal kill.

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]

HISTOPATHOLOGY / ORGAN WEIGTHS
At the interim and terminal kills organ weights were determined for the adrenal glands, brain, heart, kidneys, liver, spleen, thyroids, testes, ovaries, and uterus. The organ weight/body weight percentages were calculated.

abdominal aorta
adrenal gland (2)
bone and bone marrow (femur)
brain (3 sections including
frontal cortex and basal
ganglia, parietal cortex
and thalamus; cerebellum
and pons)
cecum
colon
duodenum
esophagus
eye
heart (with coronary vessles)
ileum
jejunum
kidneys (2)
liver
lung and mainstem bronchi
mediastinal lymph node
mesenteric lymph node
mammary gland
mandibular salivary gland
nerve (sciatic)
ovaries
pancreas
pituitary gland
prostate
seminal vesicles
skeletal muscle (rectus femoris)
skin
spinal cord (cervical)
spleen
stomach
testes with epididymides
thymus
trachea
thyroid/parathyroid
urinary bladder
uterus
gross lesions of uncertain
nature and all tissue
masses or suspect tumors and
abnormal regional lymph nodes

A blood smear (air dried/methanol fixed) was taken from the animals at the scheduled kills for possible histopathologic evaluation. Blood smears
were also taken from moribund animals starting with those after the interim kill.

Tumor incidence analysis.

Statistics:

The controls were combined, weighted for the number of samples in each, for statistical analyses. Differences between mean values were analyzed
using Dunnett’s t-test [I]. Ratios were compared using a 2x2 contingency table with Yates’ correction. A probability of p<0.05 was used as a basis to determine statistical significance. For body weight, organ weight and clinical pathology data, if a significant difference was observed between a dose and combined control group, the two controls were compared to each other using a Student’s t-test at the p<0.01 level. If the controls differed, then each control group was compared to the dose groups using Dunnett’s t-test at the p<0.05 level. Statistical analyses for tumor incidences where values of p<0.0l were considered significant was performed by using the NCI program developed by Thomas, et. al.

Reproductive indices:

reproductive performance and gesatation period

Offspring viability indices:

Pup viability was determined as gestation viability (live birth vs total birth), neonate viability (live pups Day 4 vs live pups Day 0), early lactation viability (live pups Day 14 vs live pups Day 4), and late lactation viability (live pups Day 21 vs live pups Day 14). The overall viability (live pups Day 21 vs live pups Day O) has also been calculated.

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Sporadic statistically significant differences have occurred, but were judged to be unrelated to treatment.

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Test substance intake: see table below

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Description (incidence and severity):

The decrease in total pups represents pups which were found dead, cannibalized, or sacrificed because they had escaped and could not be returned to the proper litter. The above data did not reveal any compound related effect.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The decreased mean body weight of female pups of the 500 ppm treatment level at Day 21 of lactation was the only value statistically different from the combined controls. However, the decrease was less than 10% of the control weight and was judged not to represent a compound related effect.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

For the 12 month interim kill, significantly high values for both the spleen weight and the spleen weight-body weight percentages was observed in the high dose females. The spleen weight and spleen weight-body weight percentages for the high dose males were elevated as compared to the combined control but this was not statistically different. Spleen weight and spleen weight-body weight percentages values for the high dose of both sexes were not statistically significant at the 30 month terminal kill but the values were elevated as compared to the combined values.

Gross pathological findings:

effects observed, treatment-related

Histopathological findings:

no effects observed

Description (incidence and severity):

The hemosiderosis observed in the spleens reported for the high dose females in the 12 Month Interim Pathology Report was subjectively graded to be more severe than similar lesions in the control animals, but the pathologist judged that this effect was unrelated to the test material. However, the hematology data reveal.ed a significant decrease in erythrocyte count and a significant increase in the spleen weight in the high dose females (500 ppm) at the 12-month interim kill. At this interval the apparent slight increase in hemosiderin of the spleen in the high dose females tends to corroborate the hematologic data and the increase of the spleen weight.
The histopathologic evaluation of the control and high dose animals in addition to grossly noted lesions from mid and low dose animals after the 12-month interim kill did not reveal any obvious compound related effect. The lesions observed in the spleens of the high dose females at the 12 Month interim kill were not apparent at the time of the terminal sacrifice.

sex ratio: No statistically significant differences between treated and combined controls were observed.

pub deaths: distribution of pup deaths was uniform among all 5 groups, for both the treated and the control. Therefore, it was judged that the deaths which occurred were not compound related, and the absence of gross necropsy data did not affect the scientific integrity of the study.

The tumor incidence analysis did not suggest any compound related effect,

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Reproductive effects observed:

not specified

The mean intake (mg/kg/day) of test material of the F1 generation has been calculated as the mean daily food

consumption multiplied by the intended dietary concentration divided by the mean body weight. The mean intake of test

material per animal over the course of the FI generation study was as follows.

Dietary Concentration(ppm of D&C Red No. 9)	mg/kg/day (Mean ± SE) males	mg/kg/day (Mean ± SE) females
100	5.08 + 0.41	6.36 + 0.39
200	10.02 + 0.82	12.53 + 0.80
500	25.89 + 2.12	32.42 + 2.12

mean material intake F0 generation

The food consumption values during lactation were excluded from the mean intake of test material per animal during the course of the F0 phase, because the food consumption values during this period reflected the food consumed by both the pups and the dams

Dietary Concentration(ppm of D&C Red No. 9)	mg/kg/day (Mean ± SE) males	mg/kg/day (Mean ± SE) females
100	8.3 ± 0.03	8.5 ± 0.21
200	17.4 ± 0.73	16.9 ± 0.44
500	42.5 ± 1.54	42.2 ± 1.05

Table 1: Cumulative deaths (percent) and numbers of survivors in parentheses by week in F0 rats

Week	Sex	Dose [ppm of Red No. 9]
		0 (Control 1)	0 (Control 2)	100	200	500
1 2 3 4 5 6 7 8 12	Male	0 (60) 0 (60) 0 (60) 0 (60) 0 (60) 0 (60) 0 (60) 0 (60) 0 (60)	0 (60) 0 (60) 0 (60) 0 (60) 0 (60) 0 (60) 0 (60) 0 (60) 0 (60)	0 (60) 0 (60) 0 (60) 0 (60) 0 (60) 0 (60) 0 (60) 0 (60) 0 (60)	0 (60) 0 (60) 0 (60) 0 (60) 0 (60) 0 (60) 0 (60) 0 (60) 0 (60)	0 (59) 0 (59) 0 (59) 0 (59) 0 (59) 0 (59) 0 (59) 0 (59) 0 (59)
1 2 3 4 5 6 7 8	Female	0 (60) 0 (60) 0 (60) 0 (60) 0 (60) 0 (60) 0 (60) 0 (60)	0 (60) 0 (60) 0 (60) 0 (60) 0 (60) 0 (60) 0 (60) 0 (60)	0 (60) 1.7 (59) 1.7 (59) 1.7 (59) 1.7 (59) 1.7 (59) 1.7 (59) 1.7 (59)	0 (60) 0 (60) 0 (60) 0 (60) 0 (60) 0 (60) 0 (60) 0 (60)	0 (61) 0 (61) 0 (61) 0 (61) 0 (61) 0 (61) 0 (61) 0 (61)
Day of gestation 0 6 15 21		0 (60) 0 (60) 0 (60) 0 (60)	0 (60) 0 (60) 0 (60) 0 (60)	1.7 (59) 1.7 (59) 1.7 (59) 1.7 (59)	0 (60) 0 (60) 0 (60) 0 (60)	0 (61) 0 (61) 0 (61) 0 (61)
Day of lactation 0 4 14 21		0 (60) 0 (60) 0 (60) 0 (60)	0 (60) 0 (60) 0 (60) 0 (60)	1.7 (59) 1.7 (59) 1.7 (59) 1.7 (59)	0 (60) 0 (60) 0 (60) 0 (60)	0 (61) 0 (61) 0 (61) 0 (61)

Table 2: Mean body weight [g] in F0 rats

Week	Sex	Dose [ppm of Red No. 9]
		0 (Control 1)	0 (Control 2)	100	200	500
0 1 2 3 4 5 6 7 8 12	Male	183 229 280 311 347 377 406 427 446 481	186 225 280 314 350 376 402 425 440 481	179 223 275 308 345 374 402 425 438 480	184 224 253* 311 348 373 397 422 436 482	179 223 267* 304 345 371 399 422 438 483
0 1 2 3 4 5 6 7 8	Female	147 166 183 201 218 230 242 250 254	147 166 190 204 219 231 242 249 256	148 167 191 207 220 232 244 251 258	147 169 190 206 221 231 245 250 258	146 169 187 199 216 225 240 245 256
Day of gestation 0 6 15 21		253 271 317 397	256 274 313 386	258 277 316 397	256 276 312 391	253 272 313 393
Day of lactation 0 4 14 21		300 317 335 302	291 307 323 295	299 308 324 299	297 307 325 292	295 304 322 303

*Significant at p < 0.05 as compared to controls: Dunnett’s t-test

Table 3: Pup viability

Indices [%]	Dose [ppm of Red No. 9]
	0 (Control 1)	0 (Control 2)	Combined Controls	100	200	500
Gestational viability (Live pups / total pups day 0)	98 (670 / 682)	99 (651/650)	99 (1321/1340)	98 (624/635)	99 (663/667)	99 (651/660)
Neonate viability (Day 4 / Day 0)	98  (659 / 670)	96 (626/651)	97 (1285/1321)	99 (619/624)*	99 (655/663)	99 (645/651)*
Early lactation viability (Day 14 / Day 4)	99 (649 / 659)	97 (605/626)	98 (1254/1285)	97 (599/619)	98 (642/655)	98 (631/645)
Late lactation viability (Day 21 / Day 14)	97 (629/649)	97 (584/605)	97 (1213/1254)	95 (569/599)	97 (623/642)	98 (617/631)
Overall viability (Day 21 / Day 0)	94 (629/670)	90 (584/651)	92 (1213/1321)	91 (569/624)	94 (623/663)	95 (617/651)

*Significant at p < 0.05 as compared to controls: Chi-Square test with Yates‘ correction

Table 4: Mean Pup Body Weight [g]

Day (mean)	Dose [ppm of Red No. 9]
	0 (Control 1)	0 (Control 2)	Combined Controls	100	200	500
0 4 14 21 (males) 21 (females)	6.5 10.3 23.6 36.9 35.0	6.5 10.1 24.8 36.0 34.7	6.5 10.2 24.2 36.5 34.9	6.7 10.0 23.4 35.0 32.7	6.6 10.3 23.7 34.0 32.4	6.4 10.0 22.8 34.4 32.1*

*Significant at p < 0.05 as compared to controls: Dunnett’s t-test

Table 5: Male / female pups ratio

Day (mean)	Dose [ppm of Red No. 9]
	0 (Control 1)	0 (Control 2)	Combined Controls	100	200	500
0 4 14 21	332 / 337 332 / 327 324 / 325 306 / 323	300 / 351 295 / 331 285 / 320 277 / 307	632 / 688 627 / 658 609 / 645 583 / 630	294 / 329 299 / 320 284 / 315 281 / 290	314 / 347 320 / 335 308 / 334 289 / 334	326 / 325 335 / 310 323 / 308 319 / 298

Table 6: Percent cumulative deaths and numbers of survivors in parentheses in F1 rats

Month	Sex	Dose [ppm of Red No. 9]
		0 (Control 1)	0 (Control 2)	100	200	500
0 6 12 18^C 24 30	Male	0 (71)^d 2.8 (69) 2.8 (69) 16.9 (59) 38.9 (44) 78.9 (15)	0 (70) 0 (70) 0 (70) 24.3 (53) 44.3 (39) 78.5 (15)	0 (70) 0 (70) 0 (70) 21.4 (55) 45.7 (38) 78.5 (15)	0 (70) 0 (70) 1.4 (69) 22.9 (54) 37.1 (44) 74.3 (18)	0 (70) 1.4 (69) 1.4 (69) 24.3 (53) 44.3 (39) 78.5 (15)
0 6 12 18^C 24 30	Female	0 (69)^d 0 (69) 2.9 (68) 20.3 (55) 43.5 (39) 75.4 (17)	0 (70) 2.9 (68) 2.9 (68) 21.4 (55) 51.4 (34) 74.3 (18)	0 (70) 1.4 (69) 1.4 (69) 22.9 (54) 40.0 (42) 72.9 (19)	0 (70) 1.4 (69) 5.7 (66) 25.7 (52) 51.4 (34) 70.0 (21)	0 (70) 1.4 (69) 2.8 (68) 22.9 (54) 38.6 (43) 68.6 (22)

^Cincludes interim kill

^done animal originally mis-sexed

Executive summary:

The test material, D&C Red No. 9, was administered to rats in the diet at concentrations of 100, 200 and 500 ppm for 8 weeks prior to mating. The treatment was continued during gestation and lactation. The test material was judged not to have an effect on body weight, food consumption, or fertility of the F0 generation rats. Similarly, no effect was evident on the viability or growth of F1 pups from birth to weaning. Rats from parents treated with graded dietary concentrations of D&C Red No. 9 (0, 100, 200 and 500 ppm) have been continued on treatment for 30 months after weaning. The only difference between the treated groups and the control (0 ppm) groups now judged to be related to the test material is the increase in the spleen weight in the high dose females at the 12 month interval. The apparent decreased values of red cell parameters seen at 12 months were not evident at 18 or 24 months. The gross and histopathologic evaluation and the tumor incidence analyses of the animals did not reveal any compound related effect.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

32 mg/kg bw/day

Study duration:

chronic

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

The test material, D&C Red No. 9, was administered to rats in the diet at concentrations of 100, 200 and 500 ppm (equ. to 8, 14 and 42 mg/kg bw/day) for 8 weeks prior to mating (CFTA 1982). The treatment was continued during gestation and lactation. The test material was judged not to have an effect on body weight, food consumption, or fertility of the F0 generation rats. Similarly, no effect was evident on the viability or growth of F1 pups from birth to weaning. Rats from parents treated with graded dietary concentrations of D&C Red No. 9 (0, 100, 200 and 500 ppm) have been continued on treatment for 30 months after weaning. The only difference between the treated groups and the control (0 ppm) groups now judged to be related to the test material is the increase in the spleen weight in the high dose females at the 12 month interval. The apparent decreased values of red cell parameters seen at 12 months were not evident at 18 or 24 months. The gross and histopathologic evaluation and the tumor incidence analyses of the animals did not reveal any compound related effect.

In a multi-generation reproduction study in Charles River CD rats, D & C Red #9 was fed in the diet at dosage levels of 0. 05, 0.5, 1. 5 and 5.0 mg/kg/day. Ten male and 20 female rats were used in each treated group and also as a control group. No changes considered to be related to compound were seen for parental rats or pups with respect to general behavior or appearance, body weight or survival. The fertility, gestation, viability and lactation indices of all litters were comparable for the control and treated groups. An examination of the ovaries and uteri of all dams sacrificed on day 19 of gestation in the F2c litter failed to reveal any abnormal gross anatomical changes. No unusual changes were observed in the examination of pups stillborn or dying during the study. No compound related gross or microscopic pathologic lesions were observed in any F1 or F3a rats from this study which were sacrificed and necropsied. No compound related organ weight variations were observed at sacrifice in F1 parental rats.

Effects on developmental toxicity

Description of key information

In a GLP-compliant teratology study according to OECD Guideline 414 in female rats, the test item was administered by gavage at dosage levels of 0, 3, 10, and 30 mg/kg/ day. Due to the absence of adverse developmental findings, the NOAEL for developmental toxicity was determined to be 30 mg/kg bw/day.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Feb - Aug 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

25 Jun 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt Rheinland-Pfalz

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: DEB2228074 ex Clariant
- Expiration date of the Batch: 01 May 2028
- Purity: 90.0 % area
- Physical state / appearance: solid / red

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: The stability of the test substance in deionized water over a period of 7 days at room temperature waas demonstrated before the start of the study.
- Solubility and stability of the test substance in the solvent/vehicle: Before and during administration, the preparations were kept homogeneous with a magnetic stirrer.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance preparations were prepared at the beginning of the administration period and thereafter at intervals, which took into account the period of established stability. The preparations were kept at room temperature.
- Final preparation of a solid: For the test substance preparations, the specific amount of test substance was weighed, topped up with 0.5 % CMC suspension in deionized water in a calibrated breaker and intensely mixed with a magnetic stirrer.

FORM AS APPLIED IN THE TEST (if different from that of starting material) : suspension

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: about 10 - 12 weeks
- Weight at study initiation: varied between 162.1 - 218.6 g
- Housing: individually in Polycarbonate cages type III
- Diet (e.g. ad libitum): ground Kliba maintenance diet mouse / rat "GLP", meal, supplied by Granovit AG, Kaiseraugst, Switzerland; ad libitum
- Water (e.g. ad libitum): potable tap water in water bottles; ad libitum
- Acclimation period: start of the study until first administration on GD 6

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 °C
- Humidity (%): 45 - 65 %
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 h / 12 h

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5 % suspension in deionized water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The specific amount of test substance was weighed, topped up with 0.5 % CMC suspension in deionized water in a calibrated beaker and intensely mixed with a magnetic stirrer.

VEHICLE
- Concentration in vehicle: 0, 0.03, 0.10, and 0.30 g/100 mL
- Amount of vehicle (if gavage): 10 mL/kg bw/d

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of the test substance preparations were sent three times (at the beginning (samples 1-9) and after the end of administration (reserve samples 1R-9R and samples 11-18)) to the analytical laboratory for verification of the concentrations. The samples taken for the concentrations control analyses were also used to verify the homogeneity of the samples of the low- and high-concentrations each (3 and 30 mg/kg bw/d). Three samples (one from the top, middle and bottom) were taken for each of these preparations from the preparation vessel with a magnetic stirrer running.

Details on mating procedure:

The animals were paired by the breeder ("time-mated"); the day of evidence of mating (= detection of vaginal plug / sperm) was referred to as GD 0.

Duration of treatment / exposure:

The test substance preparations were administered to the animals once a day orally by gavage, from implantation to one day prior to the expected day of parturition (GD 6 to GD 19).

Frequency of treatment:

once daily

Duration of test:

20 days

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

vehicle control

Dose / conc.:

3 mg/kg bw/day (nominal)

Remarks:

low-dose level

Dose / conc.:

10 mg/kg bw/day (nominal)

Remarks:

mid-dose level

Dose / conc.:

30 mg/kg bw/day (nominal)

Remarks:

high-dose level

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were chosen at the request of the sponsor.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily before and after treatment period (GD 0 - 5 and 20). During treatment period (GD 6 - 19) all rats were checked daily.
- Cage side observations checked: signs of morbidity, pertinent behavioral changes and / or signs of overt toxicity before administration as well as within 2 hours and within 5 hours after administration

DETAILED CLINICAL OBSERVATIONS: No
- Time schedule:

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on GD 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule: The consumption of food was recorded for the intervals GD 0-1, 1-3, 3-6, 6-8, 8-10, 10-13, 13-15, 15-17, 17-19 and 19-20.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations:

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: thyroid glands, uteri, ovaries, spleen

OTHER: hematology, clinical chemistry, thyroid hormones

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: live fetuses, dead fetuses

Fetal examinations:

- External examinations: Yes
- Soft tissue examinations: Yes
- Skeletal examinations: Yes
- Head examinations: No

Statistics:

- Simultaneous comparison of all dose groups with the control group using the DUNNETT-test (two-sided) for the hypothesis of equal means
- Pairwise comparison of each dose group with the control group using FISHER'S EXACT test (one-sided) for the hypothesis of equal proportions
- Pairwise comparison of each dose group with the control group using the WILCOXON test (one-sided) for the hypothesis of equal medians
- Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (twosided) for the hypothesis of equal medians
- Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians
- Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the equal medians

Indices:

- conception rate
- preimplantation loss
- postimplantation loss
- anogenital distance

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any female at dose levels of 3, 10 or 30 mg/kg bw/d during the entire study period.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no test substance-related or spontaneous mortalities in any females of all test groups (0, 3, 10 or 30 mg/kg bw/d).

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The mean body weights and the average body weight gain of the low-, mid- and high-dose dams (3, 10 and 30 mg/kg bw/d) were generally comparable to the concurrent control group throughout the entire study period.
The corrected body weight gain of test groups 1, 2 and 3 (3, 10 and 30 mg/kg bw/d) revealed no difference of any biological relevance to the corresponding control group. Moreover, mean carcass weights of all test groups remained unaffected by the treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The mean food consumption of the high-, mid- and low-dose dams (30, 10 and 3 mg/kg bw/d) was generally comparable to the concurrent control group throughout the entire study period.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

At gestation day 20, in dams of test groups 2 and 3 (10 and 30 mg/kg bw/d) red blood cell (RBC) counts, hemoglobin and hematocrit values were significantly decreased whereas absolute reticulocyte counts were significantly increased. Although each parameter was only slightly changed, the sum of altered red blood cell parameters was regarded as treatment-related and adverse.
Mean corpuscular hemoglobin concentration (MCHC) was significantly decreased in dams of test group 3 (30 mg/kg bw/d), but the mean was within the historical control range (MCHC 21.16-22.65 mmol/L). The same was true for significantly increased absolute and relative monocyte counts in dams of test groups 1, 2 and 3 (3, 10 and 30 mg/kg bw/d; test group 3 no significant increase) as well as decreased relative eosinophil counts in dams of test group 3 (absolute monocytes 0.08-0.18 Giga/L; relative monocytes 2.0-3.1 %, relative eosinophils 0.8 - 2.0 %). Therefore, these changes were regarded as incidental and not treatment-related.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At gestation day 20, in dams of test groups 2 and 3 (10 and 30 mg/kg bw/d) total bilirubin values were significantly increased. In combination with the changed red blood cell parameters, this alteration was regarded as treatment-related and adverse.
In dams of test group 2 (10 mg/kg bw/d) albumin values were significantly decreased, but the change was not dose-dependent. Therefore, it was regarded as incidental and not treatmentrelated.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Absolute weights:
When compared to control group 0 (= 100 %), the mean absolute weight of the spleen was significantly increased in test group 3. All other mean absolute weight parameters did not show significant differences when compared to the control group 0.

Relative organ weights
When compared to control group 0 (= 100 %), the mean relative weights of the spleen were significantly increased in test groups 2 and 3. All other mean relative weight parameters did not show significant differences when compared to the control group 0.

In test group 3, the significant increases in absolute (+20 %) and relative (+21 %) spleen weights were assessed as treatment-related.
In test group 2, the significantly increased relative spleen weight (0.224%) was within historical control range values (0.193 % - 0.227 %) and showed no histopathological correlate. Therefore, these changes were considered not treatment-related.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The spleen of 5 out of 25 animals in test group 3 and 1 out of 25 animals in test group 2 was enlarged.
All other findings occurred individually. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related findings were observed in the spleen of females of test group 3.
All other findings were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Other effects:

no effects observed

Description (incidence and severity):

In dams of test groups 1, 2 and 3 (3,10 and 30 mg/kg bw/d) no treatment-related alterations of T3, T4 and TSH levels were observed.

Pre- and post-implantation loss:

effects observed, non-treatment-related

Total litter losses by resorption:

no effects observed

Early or late resorptions:

effects observed, non-treatment-related

Dead fetuses:

no effects observed

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

The conception rate was 92% in the mid-dose group (10 mg/kg bw/d) and 100% in the control and the low- and high-dose groups (0, 3 and 30 mg/kg bw/d). With these rates, a sufficient number of pregnant females were available for the purpose of this study.

Details on maternal toxic effects:

There were no test substance-related and/or biologically relevant differences between the test groups in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and post-implantation losses, the number of resorptions and viable fetuses. This includes the statistically significant lower number of resorptions/ early resorptions, and lower value for postimplantation loss, as well as the statistically significant higher number of live fetuses in test group 2 (10 mg/kg bw/d) which was assessed as incidental. All observed differences are considered to reflect the normal range of fluctuations for animals of this strain and age.

Dose descriptor:

NOAEL

Effect level:

3 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

haematology

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The mean fetal weights of test groups 1, 2 and 3 were not influenced by the test substance and did not show any biologically relevant differences in comparison to the control group.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex distribution of the fetuses in test groups 1-3 (3, 10 and 30 mg/kg bw/d) was comparable to the control fetuses.

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No external malformations were recorded.
No external variations were recorded.
One unclassified external observation was recorded. Fused placentae were seen in one litter, each, of test groups 1 and 2 (3 and 10 mg/kg bw/d). This finding was not considered biologically relevant, since it was a single event in the respective dose group.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Skeletal malformations were recorded for one fetus, each, in test groups 0, 2 and 3 . All findings were single events in individual fetuses and no ontogenetic pattern was obvious, therefore, the observed malformations were not assessed as treatment-related and adverse. The mean values of total incidences of skeletal malformations did not differ significantly and were within the historical control data.
For all test groups, skeletal variations of different bone structures were observed, with or without effects on corresponding cartilages. The observed skeletal variations were related to several parts of fetal skeletons and appeared without a relation to dose. The overall incidences of skeletal variations were comparable to the historical control data.
The increased incidences of skeletal variations were not related to the dose and they were inside the historical control range. Therefore, they were not considered as treatment-related.
Additionally, some isolated cartilage findings without impact on the respective bony structures, which were designated as unclassified cartilage observations, occurred in all test groups. The observed unclassified cartilage findings were related to the skull, the ribs and the sternum and did not show any relation to dosing. However, the overall incidence of unclassified cartilage observations was statistically significantly increased in test group 3 (30 mg/kg bw/d) nevertheless well within the historical control range.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Soft tissue malformations occurred in two control and one fetus, each, in test group 1 and 3 (0, 3 and 30 mg/kg bw/d). These findings were not related to dose and single events in individual fetuses. The overall incidences of soft tissue malformations were comparable to those found in the historical control data.
Four soft tissue variations were detected, i.e. malpositioned carotid branch in the control group, elongated innominate in test group 1, dilated renal pelvis in all test groups and dilated ureter in test groups 0-2. The incidences of these variations were neither statistically significantly nor
dose-dependently increased in the treated groups. Except ‘elongated innominate’, which occurred once in test group 1, all of them can be found in the historical control data at comparable incidences. Therefore, they were not assessed as treatment-related.
No soft tissue unclassified observations were recorded.

Dose descriptor:

NOAEL

Effect level:

30 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: absence of adverse effects

Abnormalities:

no effects observed

Developmental effects observed:

no

Table 2: Absolute organ weights of maternal animals

Absolute weights	Females
Test group [mg/kg bw/d]	1 [3]	2 [10]	3 [30]
Spleen	99 %	106 %	120 % **

* : p ≤ 0.05, **: p ≤ 0.01

Table 3: Relative organ weights of maternal animals

Relative weights	Females
Test group [mg/kg bw/d]	1 [3]	2 [10]	3 [30]
Spleen	98 %	108 % *	121 % **

* : p ≤ 0.05, **: p ≤ 0.01

Table 4: Treatment-related findings in the spleen with incidences and gradings

	Female animals
Test group [mg/kg bw/day]	0 [0]	1 [3]	2 [10]	3 [30]
No. of dams	25	25	23	25
Spleen	25	25	23	25
Hematopoiesis, extramedullary	25	25	23	25
-         Grade 1	5	7	3	0
-         Grade 2	17	16	14	3
-         Grade 3	3	2	6	19
-         Grade 4	0	0	0	3

Table 5: Total external unclassified observations

		Test group 0 0 mg/kg bw/d	Test group 1 3 mg/kg bw/d	Test group 2 10 mg/kg bw/d	Test group 3 30 mg/kg bw/d
Litter Fetuses	N N	25 265	25 282	23 265	25 261
Fetal incidence	N [%]	0.0	1 [0.4]	1 [0.4]	0.0
Litter incidence	N [%]	0.0	1 [4.0]	1 [4.3]	0.0
Affected fetuses/litter	Mean%	0.0	0.3	0.3	0.0

N = number

Table 6: Total soft tissue malformations

		Test group 0 0 mg/kg bw/d	Test group 1 3 mg/kg bw/d	Test group 2 10 mg/kg bw/d	Test group 3 30 mg/kg bw/d
Litter Fetuses	N N	25 128	25 134	23 128	25 123
Fetal incidence	N [%]	2 [1.6]	1 [0.7]	0.0	1 [0.8]
Litter incidence	N [%]	2 [8.0]	1 [4.0]	0.0	1 [4.2]
Affected fetuses/litter	Mean%	1.6	0.8	0.0	0.6

N = number

Table 7: Total soft tissue variations

		Test group 0 0 mg/kg bw/d	Test group 1 3 mg/kg bw/d	Test group 2 10 mg/kg bw/d	Test group 3 30 mg/kg bw/d
Litter Fetuses	N N	25 128	25 134	23 128	25 123
Fetal incidence	N [%]	4 [3.1]	9 [6.7]	4 [3.1]	2 [1.6]
Litter incidence	N [%]	2 [16]	6 [24]	4 [17]	2 [8.3]
Affected fetuses/litter	Mean%	3.4	6.2	3.4	1.9

N = number

Table 8: Total skeletal malformations

		Test group 0 0 mg/kg bw/d	Test group 1 3 mg/kg bw/d	Test group 2 10 mg/kg bw/d	Test group 3 30 mg/kg bw/d
Litter Fetuses	N N	25 137	25 148	23 137	25 138
Fetal incidence	N [%]	1 [0.7]	0.0	1 [0.7]	1 [0.7]
Litter incidence	N [%]	1 [4.0]	0.0	1 [4.3]	1 [4.0]
Affected fetuses/litter	Mean%	0.8	0.0	0.9	0.8

N = number

Table 9: Total skeletal variations

		Test group 0 0 mg/kg bw/d	Test group 1 3 mg/kg bw/d	Test group 2 10 mg/kg bw/d	Test group 3 30 mg/kg bw/d
Litter Fetuses	N N	25 137	25 148	23 137	25 138
Fetal incidence	N [%]	125 [91]	143 [97]	120 [88]	130 [94]
Litter incidence	N [%]	25 [100]	25 [100]	23 [100]	25 [100]
Affected fetuses/litter	Mean%	91.4	96.9	88.2	94.1

N = number

Table 10: Occurence of statistically significantly increased fetal skeletal variations

Finding	Test group 0 [0 mg/kg bw/d]	Test group 1 [3 mg/kg bw/d]	Test group 2 [10 mg/kg bw/d]	Test group 3 [30 mg/kg bw/d]	HCD Mean % [range]
Unossified sternebra; Unchanged cartilage	0.0	4.3 **	3.8 *	1.5	3.8 [0.0 – 9.6]
Unilateral ossification of sternebra; Unchanged cartilage	0.0	2.5 *	1.4	1.6	1.0 [0.0 – 4.6]
Wavy rib	0.7	2.7	4.9 *	2.0	4.6 [0.0 – 13.3]

HCD: Historical control data

*p ≤ 0.05 (Wilcoxon-test [one-sided])

**p ≤ 0.01 (Wilcoxon-test [one-sided])

Table 11: Total unclassified cartilage observations

		Test group 0 [0 mg/kg bw/d]	Test group 1 [3 mg/kg bw/d]	Test group 2 [10 mg/kg bw/d]	Test group 3 [30 mg/kg bw/d]
Litter Fetuses	N N	25 137	25 148	23 137	25 138
Fetal incidence	N [%]	88 [64]	109 [74]	103 [75]	112 [81]
Litter incidence	N [%]	25 [100]	24 [96]	23 [100]	25 [100]
Affected fetuses/litter	Mean%	64.8	73.5	73.7	81.3*

N = number

*p ≤ 0.05 (Wilcoxon-test [one-sided])

Table 12: Total fetal malformations

		Test group 0 [0 mg/kg bw/d]	Test group 1 [3 mg/kg bw/d]	Test group 2 [10 mg/kg bw/d]	Test group 3 [30 mg/kg bw/d]
Litter Fetuses	N N	25 265	25 282	23 265	25 261
Fetal incidence	N [%]	3 [1.1]	1 [0.4]	1 [0.4]	2 [0.8]
Litter incidence	N [%]	2 [8.0]	1 [4.0]	1 [4.3]	2 [0.8]
Affected fetuses/litter	Mean%	1.2	0.4	0.4	0.7

N = number

Table 13: Total fetal variations

		Test group 0 [0 mg/kg bw/d]	Test group 1 [3 mg/kg bw/d]	Test group 2 [10 mg/kg bw/d]	Test group 3 [30 mg/kg bw/d]
Litter Fetuses	N N	25 265	25 282	23 265	25 261
Fetal incidence	N [%]	129 [49]	152 [54]	124 [47]	132 [51]
Litter incidence	N [%]	25 [100]	25 [100]	23 [100]	25 [100]
Affected fetuses/litter	Mean%	48.9	54.0*	47.4	52.5

N = number

*p ≤ 0.05 (Wilcoxon-test [one-sided])

Conclusions:

Under the conditions of this prenatal developmental toxicity study, the oral administration of the test substance to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6 - 19) caused evidence of maternal toxicity, such as a regenerative normocytic, normochromic, hemolytic anemia at doses of 10 mg/kg bw/d and above.
In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 3 mg/kg bw/d.
There were no toxicologically relevant adverse fetal findings evident. Thus, the no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 30 mg/kg bw/d, the highest tested dose.
The test substance is not teratogenic in rats.

Executive summary:

The test substance was tested for its prenatal developmental toxicity in Wistar rats. The test substance was administered as an aqueous preparation to groups of 25 time-mated female Wistar rats by gavage at doses of 3, 10 and 30 mg/kg body weight/day (mg/kg bw/d) on gestation days (GD) 6 through 19. The control group, consisting of 25 females, was dosed with

the vehicle (0.5 % Sodium carboxymethyl cellulose (CMC) suspension in deionized water) in parallel. A standard dose volume of 10 mL/kg body weight was used for each test group. At terminal sacrifice on GD 20, 23 - 25 females per group had implantation sites.

Food consumption and body weights of the animals were recorded regularly throughout the study period. The state of health of the animals was checked each day.

On GD 20, blood samples were obtained from all females by retrobulbar venous puncture following isoflurane anesthesia. After blood sampling all females were sacrificed by decapitation (under isoflurane anesthesia) and assessed by gross pathology (including organ sampling of the spleen, the thyroid glands (including parathyroid glands) as well as weight

determinations of the spleen, thyroid glands (with parathyroid glands), the unopened uterus and placentas). For each dam, corpora lutea were counted and number and distribution of implantation sites (differentiated between resorptions, live and dead fetuses) were determined. The fetuses were removed from the uterus, sexed, weighed and further investigated for

external findings. Anogenital distance measurements were conducted on all liveborn fetuses. Thereafter, one half of the fetuses of each litter were examined for soft tissue findings and the remaining fetuses for skeletal (inclusive cartilage) findings.

Results

Analytics

• The stability of the test substance in deionized water over a period of 7 days at room temperature was demonstrated.

• The homogeneous distribution of the test substance in the vehicle was shown.

• The overall correctness of the prepared concentrations was shown.

Effects

The following test substance-related adverse effects/findings were noted:

Test group 3 (30 mg/kg bw/d):

Dams

• Decreased red blood cell (RBC), hematocrit and hemoglobin values.

• Increased absolute reticulocyte counts and total bilirubin values.

• Statistically significant increase of absolute (+20%) and relative (+21%) spleen weight.

• Spleen: Hematopoiesis, extramedullary: 22 out of 25 animals (moderate to severe).

Fetuses

• No test substance-related adverse effects on fetuses.

Test group 2 (10 mg/kg bw/d):

Dams

• Decreased red blood cell (RBC), hematocrit and hemoglobin values.

• Increased absolute reticulocyte counts and total bilirubin values.

Fetuses

• No test substance-related adverse effects fetuses.

Test group 1 (3 mg/kg bw/d):

• No test substance-related adverse effects on dams, gestational parameters or fetuses.

Under the conditions of this prenatal developmental toxicity study, the oral administration of the test substance to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) caused evidence of maternal toxicity, such as a regenerative normocytic, normochromic, hemolytic anemia at doses of 10 mg/kg bw/d and above.

In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 3 mg/kg bw/d.

There were no toxicologically relevant adverse fetal findings evident. Thus, the no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 30 mg/kg bw/d, the highest tested dose.

The test substance is not teratogenic in rats.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

30 mg/kg bw/day

Study duration:

subacute

Species:

rat

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

The test substance was tested for its prenatal developmental toxicity in Wistar rats. The test substance was administered as an aqueous preparation to groups of 25 time-mated female Wistar rats by gavage at doses of 3, 10 and 30 mg/kg body weight/day (mg/kg bw/d) on gestation days (GD) 6 through 19. The control group, consisting of 25 females, was dosed with

the vehicle (0.5 % Sodium carboxymethyl cellulose (CMC) suspension in deionized water) in parallel. A standard dose volume of 10 mL/kg body weight was used for each test group. At terminal sacrifice on GD 20, 23 - 25 females per group had implantation sites.

Food consumption and body weights of the animals were recorded regularly throughout the study period. The state of health of the animals was checked each day.

On GD 20, blood samples were obtained from all females by retrobulbar venous puncture following isoflurane anesthesia. After blood sampling all females were sacrificed by decapitation (under isoflurane anesthesia) and assessed by gross pathology (including organ sampling of the spleen, the thyroid glands (including parathyroid glands) as well as weight

determinations of the spleen, thyroid glands (with parathyroid glands), the unopened uterus and placentas). For each dam, corpora lutea were counted and number and distribution of implantation sites (differentiated between resorptions, live and dead fetuses) were determined. The fetuses were removed from the uterus, sexed, weighed and further investigated for

external findings. Anogenital distance measurements were conducted on all liveborn fetuses. Thereafter, one half of the fetuses of each litter were examined for soft tissue findings and the remaining fetuses for skeletal (inclusive cartilage) findings.

Results

Analytics

• The stability of the test substance in deionized water over a period of 7 days at room temperature was demonstrated.

• The homogeneous distribution of the test substance in the vehicle was shown.

• The overall correctness of the prepared concentrations was shown.

Effects

The following test substance-related adverse effects/findings were noted:

Test group 3 (30 mg/kg bw/d):

Dams

• Decreased red blood cell (RBC), hematocrit and hemoglobin values.

• Increased absolute reticulocyte counts and total bilirubin values.

• Statistically significant increase of absolute (+20%) and relative (+21%) spleen weight.

• Spleen: Hematopoiesis, extramedullary: 22 out of 25 animals (moderate to severe).

Fetuses

• No test substance-related adverse effects on fetuses.

Test group 2 (10 mg/kg bw/d):

Dams

• Decreased red blood cell (RBC), hematocrit and hemoglobin values.

• Increased absolute reticulocyte counts and total bilirubin values.

Fetuses

• No test substance-related adverse effects fetuses.

Test group 1 (3 mg/kg bw/d):

• No test substance-related adverse effects on dams, gestational parameters or fetuses.

Under the conditions of this prenatal developmental toxicity study, the oral administration of the test substance to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) caused evidence of maternal toxicity, such as a regenerative normocytic, normochromic, hemolytic anemia at doses of 10 mg/kg bw/d and above.

In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 3 mg/kg bw/d.

There were no toxicologically relevant adverse fetal findings evident. Thus, the no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 30 mg/kg bw/d, the highest tested dose.

The test substance is not teratogenic in rats.

Justification for classification or non-classification

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

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for reproductive toxicity under Regulation (EC) No. 1272/2008.