N-(2-chloroethyl)-4-[(2,6-dichloro-4-nitrophenyl)azo]-N-ethyl-m-toluidine

Toxicity to reproduction

Administrative data

Endpoint:

one-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

None of the deviations were considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions.

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

None of the deviations were considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions.

GLP compliance:

yes

Limit test:

yes

Justification for study design:

The dose levels in this study were selected to be 0, 50, 150 and 500 mg/kg/day, based on the results of a 10-day dose range finder in which dose limiting effectswere observed at 1000 mg/kg (No mortality observed at 1000 mg/kg).

Based on the results of this dose range finder, dose levels selected for the main study were 50, 150 and 500 mg/kg.
Since no clear peak effect of occurrence of clinical signs was observed in the dose range finder, clinical observations (clinical signs and arena) were conducted and functional observations were started in the main study shortly after dosing at no specific time point, but within a similar time period after dosing for the respective animals.

Test material

Test material information

Constituent 1

Reference substance name:

N-(2-chloroethyl)-4-[(2,6-dichloro-4-nitrophenyl)azo]-N-ethyl-m-toluidine

EC Number:

264-439-1

EC Name:

N-(2-chloroethyl)-4-[(2,6-dichloro-4-nitrophenyl)azo]-N-ethyl-m-toluidine

Cas Number:

63741-10-6

Molecular formula:

C17H17N4O2Cl3

IUPAC Name:

N-(2-chloroethyl)-4-[(2,6-dichloro-4-nitrophenyl)azo]-N-ethyl-m-toluidine

Test material form:

solid: particulate/powder

Details on test material:

Anthraquinone dye, blue powder

Specific details on test material used for the study:

Test item was a brown powder with a purity of 99%.

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

Sex:

male/female

Details on test animals or test system and environmental conditions:

At initiation of dosing, males were 10 weeks old and weighed between 256 and 293 g and females were 13 weeks old and weighed between 193 and 214 g.
A health inspection was performed before the initiation of dosing.
The total number of animals used in this study was considered to be the minimum required to properly characterize the effects of the test item. This study has been designed such that it does not require an unnecessary number of animals to accomplish its objectives.
At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist.
This type of study plan was reviewed and agreed by the Laboratory Animal Welfare Officer and the Ethical Committee of Charles River Den Bosch as required by the Dutch Act on Animal Experimentation (February 1997).

The animals were allowed to acclimate to the Test Facility toxicology accommodation for 8 days prior to start of the pre-test period (females) or 8 days before the commencement of dosing (males).

A total of 40 females was selected at randomization before initiation of the pre-test phase. Any selected female without a regular estrous cycle at the end of the pre-test phase was replaced by one of the 8 additional females having regular estrous cycles. A total of 40 females with regular estrous cycles continued in the study. The supernumerary females were removed from the study and their estrous cycle results were kept in the raw data but were not reported.
Animals were assigned to groups by a computer-generated random algorithm according to body weights, with all animals within ± 20% of the sex mean. Males and females were randomized separately.

Prior to start of the pre-test period (females) or treatment period (males), each animal was identified using earmark and tattoo. Prior to the pre-test period, reserve females were numbered R1 through R8 at random by indelible marker. Any reserve female replacing an allocated female prior to treatment received identification by earmark and tattoo.
Pups were identified on postnatal day (PND) 1. They were randomized per litter and individually identified by means of subcutaneous injection of Indian ink. When general hair growth blurred the identification, the pups were identified by tattoo on the feet.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

For F0 the oral route of administration was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.
Pups (F1) will not be treated directly but could potentially be exposed to the test item in utero, via maternal milk, or from exposure to maternal urine/feces.

Details on mating procedure:

After 14 days of treatment, animals were cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

A dose control system (DCS) was used as additional check to verify the dosing procedure according to Standard Operating Procedures.

Duration of treatment / exposure:

The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week for a minimum of 28 days. Males were treated for 29 days, up to and including the day before scheduled necropsy. This included two weeks prior to mating, during the mating and post-mating. Females that delivered were treated for 50-62 days, i.e. two weeks prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver or
had a total litter loss were treated for 43-46 days.

Frequency of treatment:

Animals were dosed approximately at the same time each day with a maximum of 5 hours difference between the earliest and latest dose. The dose volume for each animal was based on the most recent body weight measurement. The doses were given using a plastic feeding tube. The dosing formulations were stirred continuously during dose administration. Pups were not treated directly but were potentially exposed to the test item in utero, via maternal milk, or from exposure to maternal urine/feces.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

Dose Concentration (mg/mL): 0

Doses / concentrations 2

Dose / conc.:

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

Remarks:

Dose Concentration (mg/mL): 10

Doses / concentrations 3

Dose / conc.:

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

Remarks:

Dose Concentration (mg/mL): 30

Doses / concentrations 4

Dose / conc.:

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

Remarks:

Dose Concentration (mg/mL): 100

No. of animals per sex per dose:

10 animals of each sex for each dose

Control animals:

yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:

Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.
For the F0 generation clinical observations were performed once daily, beginning during the first administration of the test item and lasting throughout the dosing periods up to the day prior to necropsy. During the dosing period, these observations were performed shortly after dosing at no specific time point, but within a similar time period after dosing for the respective animals (no peak effect of occurrence of clinical signs was observed in the dose range finder).
The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected in summary tables.

Oestrous cyclicity (parental animals):

Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage.
Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period.
On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrus.

Sperm parameters (parental animals):

For the testes of selected males and all males that failed to sire, additional slides of the testes were stained with PAS/ haematoxylin to examine staging of spermatogenesis.

Litter observations:

Pups were observed daily for general health/mortality. The number of live and dead pups were determined on PND 1 and daily thereafter. Pups were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.
Pups showing pain, distress or discomfort which was considered not transient in nature or is likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7). The circumstances of any death were recorded in detail.
For pups (F1) the following clinical observations were performed at least once daily for all pups. Only days on which clinical signs were present between thefirst and last litter check were given in the respective report tables.
Live pups were weighed individually on PND 1, 4, 7 and 13. Sex was externally determined for all pups on PND 1 and 4. Anogenital distance (AGD) was measured for all live pups on PND 1. The AGD was normalized to the cube root of body weight. All male pups in each litter were examined for the number of areola/nipples on PND 13. To reduce variability among the litters, on PND 4 eight pups from each litter of equal sex distribution (if possible) were selected. Blood samples were collected from two of the surplus pups (if possible from one male and one female pup). Selective elimination of pups, e.g. based upon body weight or AGD, was not done. Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable.

Postmortem examinations (parental animals):

All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs. Necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology. A veterinary pathologist, or other suitably qualified person, was available.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Red discoloration of the faeces occurred at 500 m/kg in all animals from Day 7 onwards. Orange discoloration of the tail was noted at 150 mg/kg (in all males and four females, starting on Day 19) and 500 mg/kg (in all animals, starting on Day 11). Incidentally, a few treated animals showed brown discoloration of the mouth. This discoloration was considered to reflect the color of the test item (a brown powder). Piloerection was noted in four females treated at 500 mg/kg on Days 9 and 10. Salivation seen after dosing among animals treated with the test item, in a dose-related manner, was considered to be a physiological response rather than a sign of systemic toxicity considering its slight severity and the time of occurrence (i.e. shortly after dosing).
No additional clinical signs were noted during the weekly arena observations. Any other clinical signs noted incidentally occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and showed no dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study period among animals treated with the test item. One female of the control group (no. 45) died after blood sampling on the day of scheduled necropsy. Aspiration of blood, as evidenced by the presence of alveolar blood at marked degree, with correlating dark red discoloration of the lungs recorded at necropsy was probably the cause of death for this animal.

Female no. 64 (150 mg/kg) was sacrificed at PND 3 due to total litter loss.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males treated at 500 mg/kg showed reduced body weight gain (statistically significant)throughout the treatment period, resulting in statistically significantly reduced mean body weights at Days 8 and 15 of the mating period (5% difference from control). In females at 500 mg/kg, body weight gain was reduced during the pre-mating phase (reaching statistical significance at Day 1 of the mating period) and gestation (statistically significant at Days 7-14 post-coitum). During lactation, body weight gain of 500 mg/kg females tended to be higher (not statistically significant) than that of controls. Mean body weights of 500 mg/kg females were slightly lower (up to 6%) compared to controls, but statistical significance was reached only at Day 13 of the lactation.
No treatment-related changes in body weights or body weight gain were noted in rats treated up to 150 mg/kg. An incidental, statistically significant difference noted in 50 mg/kg females (lower weight gain at Day 7 post-coitum) was considered unrelated to treatment due to the lack of a dose-related trend.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption (before and after allowance for body weight) was reduced at 500 mg/kg in females during the first week of gestation. The differences from controls (about 10-15%) were generally statistically significant. In addition, lower food consumption (before and after allowance for body weight) was noted during the first week of the pre-mating period for the 500 mg/kg females housed in one of the cages.
No treatment-related changes in food consumption were noted in males up to 500 mg/kg and in females up to 150 mg/kg. The statistically significantly lower relative food consumption noted in 150 mg/kg females between Days 0-4 post-coitum was considered an incidental finding as similar differences were not observed at later time points. In addition, a large amount of crumbled feed pellets was observed at the bottom of the cage of female no.43 in the afternoon of Day 4 of lactation. As a result, the value for food consumption over days 4-76 of lactation for this animal was affected and excluded from interpretation. Since this food spillage was a one-time event in a control animal, no importance was attached to this finding.

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):

The following changes in haematology parameters parameters (red and white blood cell parameters, number of platelets) distinguished treated animals from control animals. The differences were statistically significant unless indicated otherwise. Relative changes in mean values (or fold change) compared to the control group are indicated between parentheses.
• Lower number of red blood cells in females at 150 and 500 mg/kg (8 and 14%, respectively). In males, the number of red blood cells tended to be lower (6%) at 500 mg/kg but statistical significance was not achieved.
• Lower haemoglobin concentration in females at 150 and 500 mg/kg (6% at both dose levels). In males, haemoglobin concentration tended to be lower (4%) at 500 mg/kg but statistical significance was not achieved.
• Higher number of reticulocytes in females at 150 and 500 mg/kg (1.5x and 1.9x of control, respectively) and in males at 500 mg/kg (1.5x of control).
• Higher mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCH) in females at 500 mg/kg (14 and 9%, respectively).
• Lower (4%) mean corpuscular haemoglobin concentration (MCHC) in females at 500 mg/kg.
The statistically significant difference in MCHC (4% lower) noted in females at 50 mg/kg was considered to be unrelated to treatment due to the lack of a clear dose-related trend and corroborating changes in other red blood cell parameters.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The following statistically significant changes distinguished treated animals from control animals. Relative changes in mean values (or fold change) compared to the control group are indicated between parentheses.
• Higher (1.8x of control) mean plasma activity of alanine aminotransferase (ALAT) in females at 500 mg/kg.
• Higher total bilirubin concentration in females at 150 and 500 mg/kg (1.5x and 2.0x of control, respectively).
• Higher (16%) potassium concentration in females at 500 mg/kg.
The other statistically significant differences noted in treated animals were considered unrelated to treatment as they occurred in the absence of a dose-related trend and all group mean values were within the normal background range for rats of the same strain an age.

Thyroid hormone analyses:
Increased levels of total T4 in were observed F0-male and female rats at all dose levels. The results and the percentages of increase relative to controls are presented in the text table below:
Males Females
Dose level (mg/kg): Cont 50 150 500 Cont 50 150 500
Total T4 4.67 6.16 5.64 6.17 4.17 5.70 6.56 6.88
% Total T4 increase 32 21 32 37 57** 65**
( relative to cont)
**: P<0.01
Whereas in treated males, the increase in level of total T4 varied between 21%-32% and a dose response relationship was not apparent, a dose related increase was observed in treated females, starting at a 37% increase in the 50 mg/kg dose group and achieving levels of statistical significance for the 57% in the 150 mg/kg and 68% in the 500 mg/kg dose group.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength was not affected by treatment.
The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with a decreasing trend in activity over the duration of the test period. In the absence of a dose-related response and statistical significance, the intergroup differences noted in females (about 40% lower mean values for total movements and ambulations at 50 and 500 mg/kg) were considered to be unrelated to treatment. All mean values were within the normal range for rats of the same strain and age.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Pathomorphologic examination was performed on 80 Wistar (Han) rats (40 F0-males, 40 F0- females) which had been subjected to a combined 28-Day oral (gavage) toxicity study with the reproduction/developmental toxicity screening test with the test item DISPERSE BROWN 27.
The rats were assigned to four dose groups, each containing 10 males and 10 females. The test item was administered once daily by gavage at doses of 50, 150 and 500 mg/kg/day (dose groups 2, 3 and 4 respectively) for a minimum of 28 days. The rats of the control group 1 received the vehicle, propylene glycol, alone. At the end of the treatment period all rats from all groups were killed and subjected to complete necropsies. Histopathologic examination was performed on an extensive list of organs and tissues from five selected group 1 and 4 F0-animals as well as the kidneys and thyroid glands of F0-males and the liver and spleen of F0-animals of both sexes from selected group 2 and 3 rats and all organs with macroscopic findings from all F0 rats.
The reproductive organs were examined from all males that failed to sire and all females that failed to deliver healthy pups. Additional slides of testes were made of the 5 selected males of groups 1 and 4 and all males suspected to be infertile and stained with PAS/hematoxylin. From one male and one female pup from each litter (if possible) the thyroid gland was examined histopathologically.

There were no test item-related unscheduled deaths.

Test item-related orange discoloration was recorded for the adipose tissue of F0 males and females of all test item treated dose groups, without microscopic correlate and in skin of the tail of males at 150 and 500 mg/kg/day and females at 500 mg/kg/day. Test item-related higher spleen weights (absolute and relative to body weight in males; relative to body weight in females) were noted at 500 mg/kg/day (microscopic correlate: extramedullary hematopoiesis) and higher liver weights (relative to body weight) were noted in the 500 mg/kg/day treated males (microscopic correlate: hepatocellular hypertrophy).

Test item-related microscopic findings were present in
• Spleen: Increased incidence and severity of extramedullary hematopoiesis (up to moderate degree) and increased pigmentation (up to slight degree) in males at 500 mg/kg/day and females at 150 and 500 mg/kg/day.
• Liver: Centrilobular hepatocellular hypertrophy (minimal degree) in the liver of a few males at 500 mg/kg/day.
• Thyroid gland: An increased incidence of minimal follicular cell hypertrophy with colloid alteration (minimal-slight) in males at 500 mg/kg/day.
• Kidneys: Increased incidence and severity of hyaline droplet accumulation (up to moderate degree) in males starting at 50 mg/kg/day.
All of the microscopic findings at the recorded incidences and severities were considered tobe non-adverse.

There were no test item related microscopic alterations in the thyroid gland of the F1-pups. There were 1/10 couples at 50 mg/kg/day and 1/10 couples at 500 mg/kg/day with no offspring and total litter loss in 1/10 females at 150 mg/kg/day. For male 31 (500 mg/kg/day) moderate tubular atrophy, germ cell degeneration in the testes and reduced luminal sperm with luminal cell debris in the epididymides accounted for the lack of offspring. In the remaining two couples, histopathology did not reveal any changes in the reproductive organs that could explain the lack of healthy offspring.

Non-adverse, test item-related morphologic alterations following the administration of DISPERSE BROWN 27 at doses up to 500 mg/kg/day for a minimum of 28 days to Wistar (Han) rats, were present in spleen and adipose tissue of both sexes and liver, thyroid gland and kidneys of males.
The morphologic alterations were present in the spleen of males at 500 mg/kg/day and females at 150 and 500 mg/kg/day and consisted of an increased incidence and severity of extramedullary hematopoiesis (with correlating increased weights and hematology parameters) and increased pigmentation; in the adipose tissue orange discoloration was present in all test item treated animals (without microscopic correlate), in the liver of males at 500 mg/kg/day hepatocellular hypertrophy (with correlating increased weights), in the thyroid gland of males at 500 mg/kg/day a minor increased incidence of follicular cell hypertrophy with colloid alteration and in the kidneys of males starting at 50 mg/kg/day an increased incidence and severity of hyaline droplet accumulation.
There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item and stage aware evaluation of the testes did not show any indication for a test item-induced abnormal spermatogenesis.

Histopathological findings: neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Length and regularity of the estrous cycle were considered to be unaffected by treatment.
Most females had regular cycles, generally of four days. Three treated females had one or two irregular cycles during the pre-mating period (nos. 60, 63 and 71 of Groups 2, 3 and 4, respectively), and one (no. 60) showed extended di-estrus during pairing. These females had normal litters, except for no. 71 which was not pregnant (likely due to impaired fertility of the male she was paired with. The observed cycle abnormalities were considered to be unrelated to treatment due to their incidental occurrence and lack of a dose-related trend.

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

Two couples had no offspring: 1/10 couples at 50 mg/kg (female/male no. 53/13) and 1/10 couples at 500 mg/kg (female/male no. 71/31). The females of these couples were not pregnant despite evidence of mating. One female at 150 mg/kg (no. 64, mated with male no. 24) had total litter loss at Day 3 of lactation.
Male no. 31 (500 mg/kg) showed moderate tubular atrophy with germ cell degeneration in the testes and reduced luminal sperm with luminal cell debris in the epididymides which accounted for the lack of offspring. This animal showed degenerative spermatogenic stages. As this can be seen as a background finding in testes of rats of this age and strain and was recorded in only a single male, this was considered to be an incidental finding unrelated to treatment.

For the two other couples, no abnormalities were seen in the reproductive organs and mammary gland which could account for their lack of offspring or total litter loss. There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item, and stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis

Details on results (P0)

No reproduction toxicity was observed up to the highest dose level tested (500 mg/kg).

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

Parental

Effect level:

> 150 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

haematology

2

Key result

Dose descriptor:

NOAEL

Remarks:

Reproductive

Effect level:

>= 500 mg/kg bw/day (actual dose received)

Based on:

test mat. (total fraction)

Sex:

male/female

Basis for effect level:

clinical signs

haematology

clinical biochemistry

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs occurred among pups that were considered to be related to treatment.
The clinical signs observed incidentally remained within the range considered normal for
pups of this age, and were therefore considered to be unrelated to treatment.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

Gestation index and duration of gestation were not affected by treatment. All pregnant females had live offspring on PND 1 (gestation index 100% for all groups).
No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature
birth. No deficiencies in maternal care were observed. Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was not affected by treatment. The survival indices were 94, 100, 91 and 95% in Groups 1, 2, 3 and 4, respectively. For females nos. 51, 52, 55 (Group 2) and 62 (Group 3) the number of pups born was slightly higher than the number of implantation sites. This phenomenon is observed from time to time and is caused by normal resorption of these areas during lactation.

The mean number of living pups at first litter check (live litter size) was not affected by treatment. Live birth index (number of live offspring on PND 1 as percentage of total number of
offspring born) was not affected by treatment. The live birth indices were 98, 98, 96 and 97% in Groups 1, 2, 3 and 4, respectively.
At first litter check, two pups of Group 1 (litter nos. 41 and 44), two pups of Group 2 (litter nos. 51 and 55), five pups of Group 3 (litter nos. 63, 64, 66 and 70) and four pups of Group 4
(litter nos. 72 and 73) were found dead. This pup mortality was considered to be unrelated to treatment as the incidence showed no dose-related trend and remained within normal limits.
Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was not affected by treatment. The viability indices were 98, 98, 93
and 97% in Groups 1, 2, 3 and 4, respectively. Two pups of Group 1 (litter no. 41), three pups of Group 2 (litter nos. 55 and 59), five pups of Group 3 (litter nos. 61, 63, 64, 66 and 69) and two pups of Group 4 (litter nos. 72 and 73) went missing (presumably cannibalized) on PND 2-3 or were found dead on PND 2-4. This post-natal loss was considered to be unrelated to treatment as the incidence showed no doserelated trend and remained within the range considered normal for pups of this age. Two pups, i.e. pup no.9 of litter 44 (Group1) and pup no.2 of litter 78 (Group 4), were euthanized in extremis on PND 3 and PND 4, respectively. The observation of a purple discolored skin and abnormal breathing in pup no.9 and a cold and lean appearance and the absence of milk in its stomach were the cause for this action. These single findings were considered incidental and not related to treatment.
Female no. 64 (Group 3) had total litter loss. She had one live pup at first litter check which went missing on PND 3.

Lactation index (number of live offspring on PND 13 as percentage of number of live offspring after culling on PND 4) was not affected by treatment. The lactation indices were
100, 100, 99 and 99% in Groups 1, 2, 3 and 4, respectively. One pup of Group 3 (litter no. 61) and one pup of Group 4 (litter no. 72) went missing (presumably cannibalized) on PND 10 and PND 6, respectively. This incidental breeding loss was within normal limits and considered to be unrelated to treatment with the test item.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean body weights of male and female pups at 500 mg/kg were statistically significantly decreased at PND 13 (by 11 and 14% in male and female pups, respectively). At PND 4 and PND 7, mean body weights of these pups were about 10% lower compared to controls but the differences were not statistically significant.
Pup body weights at birth (PND 1) were considered not to be affected by treatment up to 500 mg/kg.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Male and female PND 13-15 pups at 50 and 150 mg/kg showed statistically significantly higher serum total T4 levels than pups of the control group (about 1.7x and 1.5x of control, respectively). Mean total T4 levels at 500 mg/kg were about 1.2x higher than controls (not statistically significant). It might by argued that these differences were unrelated to treatment because their magnitude decreased with increasing dose levels of the test item. However, the differences at 50 and 150 mg/kg exceeded normal background variation. Moreover, differences were consistent across sexes.
The serum TSH levels in male and female PND 13-15 pups were considered not affected by treatment. A relatively large variation of the individual TSH values was observed within some of the treated dose groups, i.e. female pups at 50 mg/kg and male and female pups at 500 mg/kg. However, a correlation with the changes in total T4 levels individual PND 13-15 male and female pups was not observed.

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

no effects observed

Description (incidence and severity):

No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, number of implantation sites, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs).

The TSH levels seemed not to be affected and no histopathological changes were observed in the thyroid glands in these pups up to treatment at 500 mg/kg. From these results, it was concluded that the increase levels of total T4 in plasma did not resulted in a (inhibitory) feedback regulation on TSH, which was supported by the absence of histopathological changes in the thyroid glands.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

No reproduction toxicity was observed up to the highest dose level tested (500 mg/kg). No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, number of implantation sites, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs).

No treatment-related changes were noted in any of the other developmental parameters investigated in this study (i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care, and the postnatal pup parameters mortality, clinical signs, anogenital distance, areola/nipple retention and macroscopic examination).

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental

Generation:

F1

Effect level:

>= 150 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Lowest effective dose / conc.:

500 mg/kg bw/day

Applicant's summary and conclusion

Conclusions:

In conclusion, based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the following No Observed Adverse Effect Levels (NOAELs) of DISPERSE BROWN 27 were established:

Parental NOAEL: 150 mg/kg, based on changes in several haematology parameters and spleen at 500 mg/kg in females (The treatment
related changes observed in the thyroid hormones were excluded in determining the NOAEL).
Reproduction NOAEL: at least 500 mg/kg.
Developmental NOAEL: 150 mg/kg, based on decreased body weight gain in male and female pups at 500 mg/kg (The treatment related changes observed in the thyroid hormones were excluded in determining the NOAEL).

Executive summary:

The objectives of this study were to determine the potential toxic effects of DISPERSE BROWN 27 when given orally by gavage for a minimum of 28 days to Wistar Han rats and to evaluate the potential to affect male and female reproductive performance such as gonadal function, mating behavior, conception, parturition and early postnatal development.

The dose levels in this study were selected to be 0, 50, 150 and 500 mg/kg/day, based on the results of a 10-day dose range finder in which dose limiting effects were observed at 1000 mg/kg.

The following parameters and end points were evaluated in this study: mortality/ moribundity, clinical signs, functional observations (5 selected animals/sex/group), body weight, food consumption, estrous cycle length and regularity, clinical pathology (5 selected animals/sex/group), serum level of thyroid hormone T4 (F0-males), gross necropsy findings, organ weights and histopathologic examinations.

In addition, the following reproduction/developmental parameters were determined: matingand fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, serum level of thyroid hormone T4 (PND 13-15 pups) and macroscopy). Accuracy, homogeneity and stability of dosing formulations were demonstrated by analyses.

Parental results

Treated rats showed red discoloration of the faeces (at 500 mg/kg) and orange discoloration of the skin of the tail (starting at 150 mg/kg) and of adipose tissue (at all dose levels). This discoloration likely represented the test item (a brown powder) and/or test item metabolite(s). In the absence of correlating microscopic alterations, this discoloration was regarded as nonadverse. Slight salivation was noted after dosing at all dose levels (in a dose-related manner), and was

considered to be a physiological response to administration of the test item.

Piloerection was incidentally seen at 500 mg/kg in females, but considered not to be toxicologically relevant based on its temporary presence. Reduced body weight gain was observed at 500 mg/kg in males throughout the treatment period and in females during the pre-mating phase and gestation. The resulting reductions in mean body weights did not exceed 6% in comparison with the concurrent controls. These small changes in body weight (gain) were considered to be non-adverse.

Females treated at 150 or 500 mg/kg showed changes (generally dose-dependent) in several haematology parameters, including reductions in the number of red blood cells and haemoglobin concentration and increases in the number of reticulocytes, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and plasma concentration of total

bilirubin. Associated findings in the spleen at these dose levels consisted of an increased incidence and severity of extramedullary haematopoiesis (a compensatory response) and pigmentation (mostly at minimal or slight degree), and a related increase in spleen weight.

There were no clinical signs indicative of hypoxia, and mean red blood cell and haemoglobin values of treated males and females remained in the normal range. The increases in plasma bilirubin and pigment in the spleen are suggestive of increased destruction of red blood cells. The combination of effects in the red blood cells and spleen in 500 mg/kg females was considered to be adverse. The red blood cell effects in 150 mg/kg females were less severe and, therefore, regarded as non-adverse. For the same reason, the red blood cell related effects noted in 500 mg/kg males (increases in number of reticulocytes and splenic extramedullary haematopoiesis and pigmentation, tendency to lower number of red blood

cells and haemoglobin concentration) were considered non-adverse.

Male rats showed an increased incidence and severity (up to moderate degree) of hyaline droplet accumulation (likely representing alpha2u-globulin, a male rat specific protein) in the kidneys starting at 50 mg/kg (with correlating increased relative renal weight at 500 mg/kg).

In the absence of indicators of renal tubular damage, this renal change was regarded as nonadverse.Centrilobular hepatocellular hypertrophy (at minimal degree) was observed in a few males at 500 mg/kg (with correlating increased relative liver weight). In the absence of any degenerative or inflammatory changes, these treatment-related hepatic findings were considered to be non-adverse.

Increased levels of total T4 in were observed F0-male and female rats at all dose levels. Whereas in treated males, the increases in level of total T4 varied between 21%-32% and a dose response relationship was not apparent, the increases in level of total T4 in treated females were 37%, 57% and 68% in the 37% increase for the 50 mg/kg, 150 mg/kg and 500

mg/kg dose groups respectively, in comparison with controls. No corroborating morphological changes were observed in the thyroid gland in males and females. The slight increased incidence of follicular cell hypertrophy and colloid alteration observed in the thyroid gland in males at 500 mg/kg showed no dose relationship and, also based on the incidence and severity, these changes were considered to be non-adverse. Any conclusion on the adversity of the changes in total T4 could not be evaluated from the results obtained in this study.

Minor changes in clinical biochemistry parameters at 500 mg/kg in females (higher mean plasma levels of alanine aminotransferase and potassium) were considered non-adverse as they remained within normal limits and occurred without corroborating changes in other endpoints.

Reproductive results

No reproduction toxicity was observed up to the highest dose level tested (500 mg/kg).

Developmental results

Body weight gain of male and female pups was reduced at 500 mg/kg, resulting in 11% (males) – 14% (females) lower mean body weights at PND 13. At birth, pup body weights showed no treatment-related changes. Treatment-related, statistically significantly higher mean serum T4 levels (exceeding the historical control range) were noted at 50 and 150 mg/kg in male and female PND 13-15 pups (about 1.7x and 1.5x of control, respectively). Mean T4 levels at 500 mg/kg were about 1.2x higher compared to controls (not statistically significant and within normal limits). The TSH levels seemed not to be affected and no histopathological changes were observed in the thyroid glands in these pups up to treatment at 500 mg/kg. From these results, it was concluded that the increase levels of total T4 in plasma did not resulted in a (inhibitory) feedback regulation on TSH, which was supported by the absence of histopathological changes in the thyroid glands. Therefore, any conclusion on the adversity of the changes in total T4 could not be evaluated from the results obtained in this study.