Reaction mass of 2,4-bis(xylylazo)resorcinol and 2,4-bis[(4-dodecylphenyl)azo]resorcinol and 2-[(4-dodecylphenyl)azo]-4-(2,4-xylylazo)resorcinol

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

The No Observed Adverse Effect Level (NOAEL) for reproductive / developmental toxicity was considered to be 100 mg/kg/day, based on the increase in gestation length observed at 300 and 1000 mg/kg/day and the decrease in gestation index and viability indices when compared to Control.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 November 2018 to 29 July 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

OECD 422 Guideline for testing of chemicals adopted 29.07.16: Combined repeated dose toxicity study with the reproduction/developmental toxicity screening test.

Deviations:

yes

Remarks:

See "Any other information" for details

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

Method: Oral gavage was used as the administration route as recommended by the OECD 422 guideline, in order to deliver accurate doses. In addition, oral ingestion is a possible route of human exposure to the test item.
Dose levels: The doses were chosen based on the preliminary results obtained in non-GLP Study RN78JF 14-day Oral (Gavage) Dose-Range Toxicity Study for OECD 422 conducted at Envigo CRS, S.A.U.
As no toxicity was obtained at the administered doses in RN78JF, then:
- The high dose was selected as no toxicity was observed in the preliminary study at 1000 mg/kg/day and considering it as a limit dose to be tested.
- Intermediate and low dose levels were selected considering approximately a 3-fold interval between doses.

Specific details on test material used for the study:

No further details specified in the study report.

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Recognized by international guidelines as a recommended test system.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Species: Hannover Wistar Rat (HsdHan®:WIST)
Supplier: Envigo RMS, S.L.
Breeder: Envigo RMS B.V., Kreuzelweg 53, 5961 NM Horst, Netherlands
Total number of animals ordered 44: males and 48 females
Total number of animals in the study: 40 males and 40 females (80 in total). In addition, 4 spare males and 8 spare females. Each group started with 10 males and 10 females per group.
Age (at treatment start): Males: 9 to 10 weeks; Females: 10 to 12 weeks
Body-weight range (at treatment start): Males: 288-347 g; Females: 192-227 g

Identification
- Acclimatization: F0 => Cage card and ear tattoo
- Treatment: F0 => Cage card and individual ear tattoo; F1 offspring – Back mark (animals were observed daily together with pup status and the back mark was checked)
Randomization: Method based on the similarity of mean body weights among groups. Before treatment starts and after the estrous cycle examination done during pre-treatment, females number 74 and 103 that did not show 4-5 day cycles were replaced with those females available from the spare group that most closely resemble their body weight.
Allocation: The animals were allocated at random to four treatment groups. Spare animals were used and/or animals were exchanged. The rejected animals took no further part in the study after commencement of treatment.

Animal Care
Acclimatization: From five days after arrival and pre-treatment start. After acclimatization period, the animals were subjected to a pre-test period.
Veterinary examination: During acclimatization (at arrival), the animals were examined by a veterinary surgeon. Only animals without any visible signs of illness were used for treatment. Before treatment start, the animals were not inspected due to an oversight and as a consequence, a veterinary inspection was done on treatment day 2 in order to confirm their health status.
Additional inspections were done to those animals showing signs of toxicity before their sacrifice in order to determine their health status.
Environmental enrichment program: Different types of material specific to this species were supplied to reduce stress, enhance wellbeing and improve behavior.

Husbandry
Room number: 3 and 111
Conditions: Optimum hygienic conditions behind a barrier system: air-conditioned with a minimum of 15-20 air changes per hour, and continuously monitored environment with ranges for temperature of 20-24 ºC and for relative humidity between 35 and 60%. 12 hours fluorescent light/12 hours dark.
Accommodation: Cages with standard, granulated, S8-15 sawdust bedding (J. Rettenmaier & Söhne)
Premating period (maximum 5 animals/cage) Makrolon type-IV cages
Mating period (one male and one female/cage) Makrolon type-III cages
Postmating, gestation and lactation periods (individual) Makrolon type-III cages
Diet: Pelleted standard Teklad 2014C rat/mouse maintenance diet ad libitum (supplied by Envigo RMS, S.L.). Pelleted standard Teklad 2018C rat/mouse maintenance diet (supplied by Envigo RMS, S.L.) ad libitum, for lactating females and pups (until sacrifice).
Water: Tap water in bottles ad libitum.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Vehicle
Identification: Corn oil
Supplier: Sigma-Aldrich
Reference: C8267
Batch number: MKBW9504V MKCG3257
Expiry date (retest date): 11 January 2019 14 December 2020, 16 January 2021 (2 years from the date of opening of the container)
Storage conditions: Stored at ambient conditions (20 ± 5ºC)
Safety precautions: Routine hygienic procedures (nitrile gloves, goggles, facemask)

Test Item Preparation and Analysis
Formulation
Group 1: Vehicle control
Group 2: Solvent Yellow 175; 20 mg/mL
Group 3: Solvent Yellow 175; 60 mg/mL
Group 4: Solvent Yellow 175; 200 mg/mL

Dose volume: 5 mL/kg/day
Concentration range to be validated: 1 mg/mL to 200 mg/mL
Frequency of dose formulation preparation: No more than 7 days between preparation and administration based on stability data.
Storage of dose formulations: At room temperature (20 ± 5 ºC) and in the absence of light.
Stability of dose formulations: At room temperature (20 ± 5 ºC) and in the absence of light for 8 days.
Safety precautions: Safety precautions were taken according to the test item hazard class and the results of the risk assessment

Method of Preparation
Aliquoting of test item
1) The container with the test item (in its original container) was immerse in a water bath, and the test item was heated to 70-80 ºC until it become liquid.
2) The test item only allows two heating cycles.
3) The density of the test item was calculated.
4) The test item was aliquoted for each preparation day in single-use containers.
5) Aliquots were stored at 20 ± 5 ºC until use.

Formulation preparation (High Concentration / Dose)
1) The aliquot containing the test item (weighed in advance and stored under the same conditions as the test item) was taken.
2) The required amount of corn oil was added at room temperature.
3) The formulation was heated to 60-70 ºC until the mixture was homogenous (the time spent, temperature and the equipment used were recorded in the raw data).
4) The formulation net weight was recorded.
5) Formulation was mixed using a magnetic stirrer during 10 minutes. A sample, when necessary, was taken at this point.
6) The rest of the formulations were prepared by dilution in a descending order of concentration.

Formulation dilution:
1) The requested volume of the most concentrated formulation was taken and diluted with vehicle to obtain the final volume and the desired concentration.
2) Formulations were then mixed using a magnetic stirrer during 10 minutes. A sample, when necessary, was taken at this point.
3) Formulations were maintained under agitation before (in Animal House) and during administration in order to assure a homogenous suspension.
When dose formulations were prepared to be administered over several days, the stock formulation was mixed for 10 minutes at room temperature before dividing it into the aliquots required.
Formulations or aliquots of dose formulations were stored at room temperature (20 ± 5 ºC).
NOTE: test item and vehicle density were not taken into account for formulations preparation.

Details on mating procedure:

Paired for mating: After minimum of 2 weeks of treatment.
Male/female ratio: 1:1
Duration of pairing: Up to 4 days
Daily checks for evidence of mating: Ejected copulation plugs. Sperm within vaginal smear.
Day 0 of gestation: When positive evidence of mating detected.
Male/female separation: Day when mating evidence detected.
Pre-coital interval: Calculated for each female as time between first pairing and evidence of mating.
Females number 76 at 0 mg/kg/day and 87 at 100 mg/kg/day that did not give birth were sacrificed 24-26 days after the last day of the mating period.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Vehicle
Identification: Corn oil
Supplier: Sigma-Aldrich
Reference: C8267
Batch number: MKBW9504V MKCG3257
Expiry date (retest date): 11 January 2019 14 December 2020, 16 January 2021 (2 years from the date of opening of the container)
Storage conditions: Stored at ambient conditions (20 ± 5ºC)
Safety precautions: Routine hygienic procedures (nitrile gloves, goggles, facemask)

Test Item Preparation and Analysis
Formulation
Group 1: Vehicle control
Group 2: Solvent Yellow 175; 20 mg/mL
Group 3: Solvent Yellow 175; 60 mg/mL
Group 4: Solvent Yellow 175; 200 mg/mL

Dose volume: 5 mL/kg/day
Concentration range to be validated: 1 mg/mL to 200 mg/mL
Frequency of dose formulation preparation: No more than 7 days between preparation and administration based on stability data.
Storage of dose formulations: At room temperature (20 ± 5 ºC) and in the absence of light.
Stability of dose formulations: At room temperature (20 ± 5 ºC) and in the absence of light for 8 days.
Safety precautions: Safety precautions were taken according to the test item hazard class and the results of the risk assessment

Method of Preparation
Aliquoting of test item
1) The container with the test item (in its original container) was immerse in a water bath, and the test item was heated to 70-80 ºC until it become liquid.
2) The test item only allows two heating cycles.
3) The density of the test item was calculated.
4) The test item was aliquoted for each preparation day in single-use containers.
5) Aliquots were stored at 20 ± 5 ºC until use.

Formulation preparation (High Concentration / Dose)
1) The aliquot containing the test item (weighed in advance and stored under the same conditions as the test item) was taken.
2) The required amount of corn oil was added at room temperature.
3) The formulation was heated to 60-70 ºC until the mixture was homogenous (the time spent, temperature and the equipment used were recorded in the raw data).
4) The formulation net weight was recorded.
5) Formulation was mixed using a magnetic stirrer during 10 minutes. A sample, when necessary, was taken at this point.
6) The rest of the formulations were prepared by dilution in a descending order of concentration.

Formulation dilution:
1) The requested volume of the most concentrated formulation was taken and diluted with vehicle to obtain the final volume and the desired concentration.
2) Formulations were then mixed using a magnetic stirrer during 10 minutes. A sample, when necessary, was taken at this point.
3) Formulations were maintained under agitation before (in Animal House) and during administration in order to assure a homogenous suspension.
When dose formulations were prepared to be administered over several days, the stock formulation was mixed for 10 minutes at room temperature before dividing it into the aliquots required.
Formulations or aliquots of dose formulations were stored at room temperature (20 ± 5 ºC).
NOTE: test item and vehicle density were not taken into account for formulations preparation.

Duration of treatment / exposure:

Males: 2 weeks before pairing up to necropsy after a minimum of 5 weeks
Females: 2 weeks before pairing, then throughout pairing and gestation until days 13-15 of lactation (until the day before sacrifice)

Frequency of treatment:

Once daily
- F0 males: Two weeks prior to mating start until the day before sacrifice (for five weeks of dosing). They were then killed.
- F0 females: Two weeks prior to mating start until day 13/15 of lactation, including the day before sacrifice.
- F1: Potential indirect exposure in utero and through the milk during lactation

Details on study schedule:

Animals mated at:
Males 11-12 weeks
Females 12-13 weeks

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

20 animals per dose group (10 male/10 female)

Control animals:

yes, concurrent vehicle

Details on study design:

Dose levels The doses were chosen based on the preliminary results obtained in non-GLP Study RN78JF 14-day Oral (Gavage) Dose-Range Toxicity Study for OECD 422 conducted at Envigo CRS, S.A.U.
As no toxicity was obtained at the administered doses in RN78JF, then:
- The high dose was selected as no toxicity was observed in the preliminary study at 1000 mg/kg/day and considering it as a limit dose to be tested.
- Intermediate and low dose levels were selected considering approximately a 3-fold interval between doses.

Positive control:

Not required for this study

Parental animals: Observations and examinations:

Serial Observations/Measurements
The observations listed below were recorded.
Additional observations and examinations were performed on animals judged to be in extremis, or showing signs of ill health or unusual findings.

Viability / Mortality / Cage-side Observations
Animals and their cages: Visually inspected twice daily for evidence of reaction to treatment or ill health.
Blood sampling for hematology or blood chemistry analysis were not performed on animals that died or were sacrificed prematurely.

Body Weight
Detailed in table for. See “any other information” for details.

Food Consumption
Detailed in table for. See “any other information” for details.

Clinical Signs
Detailed in table for. See “any other information” for details.

Sensory Reactivity and Grip Strength
Detailed in table for. See “any other information” for details.

Motor Activity
Detailed in table for. See “any other information” for details.

In-life Sampling and Analysis
Hematology, Peripheral Blood
Conditions: Males and females were deprived of food from 6-8 hours before blood extraction. Pups were removed from the dam the day before this procedure.
Samples were collected under light general anesthesia.
Anesthetic: Isoflurane.
Sample site: Retro-orbital sinus
Anticoagulant/Sample volume: EDTA/0.5 mL; Citrate/0.5 mL
Routine hematology parameters are measured by the Advia 120. Coagulation parameters are measured using a STA Compact.
All samples were examined for the following characteristics, when possible:
Using EDTA as anticoagulant
Hematocrit (Hct); Hemoglobin concentration (Hb); Erythrocyte count (RBC); Absolute reticulocyte count (Retic); Total leucocyte count (WBC) Differential leucocyte count (N: neutrophils, L: lymphocytes, E: eosinophils, B: basophils, M: monocytes, LUC: large unstained cells); Platelet count (Plt); Mean cell haemoglobin (MCH); Mean cell volume (MCV); Mean cell hemoglobin concentration(MCHC)
Using citrate as anticoagulant
Prothrombin time (SPT); Activated partial thromboplastin time (SAPT)

Blood Chemistry
Conditions: Males and females were deprived of food from 6-8 hours before blood extraction.
Samples collected under light general anesthesia.
Anesthetic: Isoflurane
Sample site: Retro-orbital sinus
Anticoagulant/Sample volume: Lithium heparin/0.8 mL
Routine biochemistry parameters were measured using the Cobas 6000 analyzer.
Electrophoretic parameters were performed by HYDRASIS LC from Sebia.
The albumin/globulin ratio was derived from the total protein value from the Cobas 6000 and the a lbumin value generated by the HYDRASYS LC from Sebia.
All samples were examined for the following characteristics:
Using lithium heparin as anticoagulant
Alkaline phosphatase (ALP); Alanine amino-transferase (ALT); Aspartate amino-transferase (AST); Glucose (Gluc); Bilirubin – total (Bili); Cholesterol – total (Chol); HDL; LDL; Triglycerides (Trig); Creatinine (Creat); Creatine kinase (CK); Urea; Total protein (Total Prot); Albumin (Alb); Albumin/globulin ratio; Protein electrophoretogram; Sodium (Na); Potassium (K); Chloride (Cl); Calcium (Ca); Phosphorus (Phos); Bile acids (Bi Ac)

Oestrous cyclicity (parental animals):

Dry smears Using inoculation loops during the following phases:
-For 14 days before treatment (all females including spares).
-Daily from the beginning of treatment period until evidence of mating.
-On the day of necropsy
A cotton swab impregnated in distilled water was used in order to check the evidence of mating during the mating period.

Sperm parameters (parental animals):

Not examined

Litter observations:

Clinical observations: Observed 24 hours after the considered birth and then daily for evidence of ill-health or reaction to maternal treatment
Litter size: Daily from Day 1-13 of age
Sex ratio: Days 1, 4, 7 and 13 of age
Individual offspring body weights: Days 1, 4, 7 and 13 of age
Ano-genital distance: Day 1 - all F1 offspring
Nipple/areolae count: Day 13 of age - male offspring.

Postmortem examinations (parental animals):

Necropsy
F0 Males: After final investigations completed (after 5 weeks of treatment)
F0 Females failing to produce viable litter (not pregnant): Day 25-26 after mating
F0 Females whose litters die before Day 13: On or after day last offspring dies
Females killed at Termination: Day 14-16 of lactation
Note: All animals that exhibit signs of undue stress or discomfort as judged by the animal welfare officer and study director were sacrificed immediately for ethical reasons. The Sponsor was informed. The reason for sacrifice is included in the report.

Method of Sacrifice
All surviving F0 animals: By intraperitoneal injection of sodium pentobarbital. Each animal will be subsequently exsanguinated.

Organ Weights
Detailed in table form. See “Any other information”-Pathological parameters.
Data collection: For bilateral organs, left and right organs were weighed together. Organ weights were not recorded for animals sacrificed prematurely.

Macroscopic Pathology
Detailed in table form. See “Any other information”-Pathological parameters.
Blood sampling required: specific details are included in this section as regards animals from which samples are required for the analysis of thyroid hormone levels (see Thyroid Hormone Analysis). Blood samples are also required at termination from specific adult animals for the evaluation of hematology and blood chemistry parameters and details relating to these examinations are included in Hematology, Peripheral Blood and Blood Chemistry of report.
Premature decedents (Sacrificed for Welfare Reasons): females 90 and 91 at 300 mg/kg/day and females 101 and 109 at 1000 mg/kg/day
Checks: retained tissues

Scheduled termination
F0 animals (five males and five surviving lactating females with a surviving litter per group): Blood sampling required from all males and females (see Thyroid Hormone Analysis).
Complete necropsy: all animals.
Checks: retained tissues.
Number of uterine implantation sites counted and checked.
Remaining F0 males and females / Females not pregnant/ fail to litter and litter death: Limited list.
Checks: retained tissues.
Number of uterine implantation sites counted and checked.
For females whose litter dies or those in which the whole litter needs was sacrificed because female was not taking maternal care: including evaluation of mammary tissue.

Fixation
Detailed in table form. See “Any other information”-Pathological parameters.
Fixatives: Standard - 10% Neutral Buffered Formalin. Testes and epididymides: Initially in modified Davidson’s fixative. Eyes: Davidson’s fixative.

Histology
Detailed in table form. See “Any other information”-Pathological parameters.
Processing - Full list: All adult animals sacrificed or dying prematurely (sacrificed for welfare reasons) from groups 1, 3 and 4. The five males selected in Groups 1 and 4 and five lactating females with a surviving litter selected in Groups 1 and 3 and the two females sacrificed at the end of the study in Group 4, at scheduled termination.
Processing – Abnormalities only: All adult animals
Routine staining: Stained with hematoxylin and eosin, except testes which are also stained with periodic acid-Schiff, according to Envigo CRS, S.A.U. internal SOPs.
Special staining: None
Extension of Initial Examination: The five F0 males selected in Groups 2 and 3: thyroids, kidneys and thymus. The five F0 lactating females with a surviving litter selected in Group 2: thymus, cecum and stomach.

Postmortem examinations (offspring):

Necropsy
F1 Offspring: Selected offspring for thyroid hormone analysis – Day 4 of age (two females per litter where possible)
Scheduled sacrifice - Day 13-15 of age

Method of Sacrifice
Offspring Selected for thyroid hormone sampling: Cardiac puncture. Death was assured with sodium pentobarbital injection.
All other offspring: injection of sodium pentobarbital.

Premature decedents (Sacrificed for Welfare Reasons)
Offspring: External examination with an assessment of stomach for milk content

Scheduled termination
F1 offspring on Day 4 of age: Blood sampling required from two females per litter where possible. No macroscopic examination was done.
F1 offspring on Day 13 of age: Blood sampling required from two males and two females per litter, where possible. Thyroid gland retained from one male and one female in each litter where possible. When the number of Day 13 pups available for hormone sampling was insufficient, priority was given to hormone sampling, with the sample for T4 given first priority. All animals were subjected to an external macroscopic examination; particular attention was paid to the external genitalia.

Statistics:

Data Types
The following data types were analyzed at each time point separately:
- Body weight, using absolute weights
- Food consumption, over appropriate study periods
- Blood chemistry and hematology (including coagulation)
- Hormone analyses
- Organ weights, both absolute and adjusted for terminal body weight
- Grip strength and motor activity
- Estrous cycles and pre-coital interval
- Mating performance and fertility
- Gestation length
- Litter data (litter size, offspring survival, offspring body weight) ano-genital distance, average for each litter adjusted for litter average pup body weight
- Number of nipples/areolae in male pups counted on post natal Day 13

Reproductive indices:

Mating performance and fertility
Group values calculated for males and females separately for the following:
Percentage mating
Conception rate
Fertility index
Gestation length
Gestation index
Litter size

Offspring viability indices:

Survival indices (%) Individual litter values calculated for:
Post-implantation survival index
Live birth index
Viability index
Lactation index
Sex ratio

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were no other signs of evident toxicity based on clinical signs in the surviving animals. Dark feces and yellow urine were considered related to the test item color.
Salivation recorded during the administration period was related to taste aversion (from gavage dosing)

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

No mortality was recorded among males. However, two females at 300 mg/kg/day and another two at 1000 mg/kg/day were sacrificed for welfare reasons between gestation days 22 and 23, showing signs of difficulty during parturition. In addition, one female at 100 mg/kg/day, three females at 300 mg/kg/day and six females at 1000 mg/kg/day were sacrificed as they lost their corresponding litters during late gestation and early lactation phases.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No effects were observed in body weight.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No effects were observed in food consumption.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no effects on coagulation. The decrease observed in mean hematocrit, hemoglobin, red blood cells and the corresponding increase in reticulocytes observed in the test-item-administered males was considered not relevant based on the fact that they were within the historical control data and/or there was no dose-effect relation.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no relevant effects on biochemistry, considering that all statistically significant mean values observed in biochemistry were within the historical control data.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no relevant effects on behavioral parameters (sensory reactivity, grip strength and motor activity).

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):

Changes that were considered to be related to treatment with the test item were seen in male thyroids and kidneys, female stomach and cecum, and thymus of both males and females.
Thyroid glands: Minimal hypertrophy of follicular cells was seen in three of the examined males given 1000 mg/kg/day, in one male given 300 mg/kg and in one male given 100 mg/kg.
Kidneys: An increased incidence and severity of hyaline droplet accumulation was detected in the kidneys of males given 1000 mg/kg/day compared with controls.
Stomach: A minimal focal erosion of the glandular stomach was seen in one female given 1000 mg/kg.
Cecum: Moderate regenerative hyperplasia of the crypts along with minimal apoptosis were seen in one female given 1000 mg/kg.
Thymus: Minimally to slightly decreased cortical/medullary cellularity was noted in the thymus of some of the examined males given 300 or 1000 mg/kg/day.
This change was also seen in all treated female groups, with a higher incidence and severity at 1000 mg/kg (minimal to severe) compared with the corresponding males and lower dose females. At 100 or 300 mg/kg, it was of minimal severity. In one of the examined females given 100 mg/kg where a smaller thymus was seen at necropsy, no tissue was present at microscopic examination.
In the single female given 1000 mg/kg where no decreased cellularity was detected, there was a minimal degree of cortical/medullary lymphocyte apoptosis.
Findings of an Uncertain Relationship to Treatment: Changes that were of unclear relationship to treatment with the test item were seen in female kidneys.
Kidneys: One of the examined females receiving 1000 mg/kg showed marked multifocal acute/subacute necrosis of cortical and, to a lesser extent, medullary tubules, along with marked multifocal tubular basophilia.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

All other histological findings were considered to be incidental and unrelated to the test item.
A minimal increase in adipocytes was seen in the bone marrow of one of the examined males given 1000 mg/kg/day compared with controls. However, given its isolated occurrence and in the lack of changes in the spleen, it was considered to be most probably incidental, as a result of normal individual variation.
An increased mucification of the cervix and the vagina occurred in a single of the examined females, which was given 300 mg/kg. This correlated with increased organ weights of this specific animal, and was considered to be incidental.
In the testes, seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage specific abnormalities were noted in the examined males treated at 1000 mg/kg/day.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Before treatment start, all females showed regular cycles. However, one female (number 81) at 100 mg/kg/day and two females at 1000 mg/kg/day (numbers 117 and 109) showed irregular cycles during treatment. Given the frequency observed, it is considered not to have any toxicological relevancy.

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

Mating performance was 100% in all test-item groups and conception and fertility were 90% at 0 and 100 mg/kg/day and 100% at 300 and 1000 mg/kg/day. This slight difference is not considered related with the test item, as it was recorded in the Control and low dose groups.

No mortality was recorded among males. However, two females at 300 mg/kg/day and another two at 1000 mg/kg/day were sacrificed for welfare reasons between gestation days 22 and 23, showing signs of difficulty during parturition. In addition, one female at 100 mg/kg/day, three females at 300 mg/kg/day and six females at 1000 mg/kg/day were sacrificed as they lost their corresponding litters during late gestation and early lactation phases.
There were no other signs of evident toxicity based on clinical signs in the surviving animals or relevant effects on behavioral parameters (sensory reactivity, grip strength and motor activity). Dark feces and yellow urine were considered related to the test item color.
Salivation recorded during the administration period was related to taste aversion (from gavage dosing).
No effects were observed in body weight or food consumption.
There were no effects on coagulation or no relevant effects on biochemistry, considering that all statistically significant mean values observed in biochemistry were within the historical control data. The decrease observed in mean hematocrit, hemoglobin, red blood cells and the corresponding increase in reticulocytes observed in the test-item-administered males was considered not relevant based on the fact that they were within the historical control data and/or there was no dose-effect relation.
In females receiving 300 or 1000 mg/kg/day, a direct toxic effect of the test item was observed in the stomach and occasionally in the duodenum and cecum and was considered to have been the main factor contributing to the unscheduled deaths.
Regarding T4 concentrations in adult males, statistically significant lower mean values were observed in the test-item-administered groups when compared to Control. Although these values were within the historical control data, minimal follicular cell hypertrophy was observed in all test-item-treated male groups, in correlation with the increased thyroid and parathyroid weights in males compared to Controls, with a higher incidence at 1000 mg/kg.
Given the concomitant increase in liver weights, follicular cell hypertrophy was most probably compensatory, secondary to a test-item-related non-adverse hepatic enzyme induction and clearance of thyroid hormones. The fact that no centrilobular hypertrophy suggestive of enzyme induction was seen in the histological examination does not rule out its occurrence.
The minor increase in hyaline droplets in the kidneys of males given 1000 mg/kg/day is consistent with treatment-related accumulation of α-2u-globulin, a common finding in untreated male rats. Hyaline droplet accumulation is specific to the male rat and is not generally considered to be significant in man. In this study, this was not accompanied by any other histologic features of pathology, and was therefore considered to be non-adverse.
The kidneys of one female treated at 1000 mg/kg/day showed histological changes which correlated with gross pallor. The single case of marked tubular necrosis in a high dose female (1000 mg/kg/day) is of unclear pathogenesis and its relation with the treatment cannot be completely ruled out.
There were minimal to severe treatment-related changes in the thymus of both sexes at 300 or 1000 mg/kg/day, with a higher incidence and severity in females. They correlated with gross findings at necropsy and/or lower organ weights recorded in males, and were interpreted as stress-related. In this study, other findings indicative of stress were occasionally found in decedent females treated at 300 or 1000 mg/kg/day (spleen, lymph nodes and pancreas). A minimal degree of decreased lymphocyte cellularity in the thymus can be normally seen among pregnant/lactating females. Thus, the minimal changes seen in two females given 100 mg/kg, in the absence of concurrent changes in the stomach or cecum, were considered to be a result of normal individual variation. A single female given 100 mg/kg had a small thymus. However, no thymic tissue was found at histological examination.
The other organ weight changes reported at the end of treatment (female brain and oviducts/uterus/cervix) did not have a correlate at histopathology. The increased weight in the reproductive organs of females given 300 mg/kg was explained by the incidental increase in cervical mucification of a single female. The increase in epididymides (mainly at 1000 mg/kg/day) was considered not relevant, taking into consideration that there were no histopathology findings corroborating it and as mean values are within the historical control data.
In the testes, seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage-specific abnormalities were noted in the examined males treated at 1000 mg/kg/day.
Estrous cycles (during treatment and at termination) and reproductive parameters of pre-coital interval, mating performance, conception or fertility were unaffected by treatment.
The duration of the gestation length was increased in the animals administered the test item as well as the gestation index was reduced in a dose relation, when compared with the Control group (mainly at 300 and 1000 mg/kg/day).
The number of live-born fetuses was reduced in a dose-related manner, as was the gestation index, in which the mean values of 300 and 1000 mg/kg/day were statistically significant.

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

histopathology: non-neoplastic

Key result

Critical effects observed:

not specified

Lowest effective dose / conc.:

100 mg/kg bw/day (nominal)

System:

gastrointestinal tract

Organ:

oesophagus

parathyroid gland

stomach

thymus

thyroid gland

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no clinical signs observed among the F1 litters that could be clearly attributable to parental treatment with Solvent Yellow 175.
Clinical signs recorded in the offspring were related with the lack of maternal care (as little or no milk present in the stomach or pallor), not due to a direct test-item effect in pups.
At 1000 mg/kg/day, thinness of pup 100-1M was recorded from day 3 onwards.
All pups sacrificed prematurely during lactation phase showed little or no milk present in the stomach and some of them were partially cannibalized.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no effect in the offspring body weight.

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:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

effects observed, non-treatment-related

Description (incidence and severity):

There was no effect in the mean ano-genital distance adjusted to body weight in males.
However, in female offspring, there was a dose-related increase in the ano-genital distance in test-item groups with respect to Control; it was statistically significant at 300 and 1000 mg/kg/day. In this case and within the confines of this study, it is considered not relevant, as mean values are within those generally observed within the Historical Control Data.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

There were no effects in male nipples.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no macroscopic findings attributable to maternal treatment with Solvent Yellow 175 in the offspring that died prior to the scheduled termination or among the offspring killed on days 4 or 13-15 of age.
The observations recorded in the offspring sacrificed after the sacrifice of the corresponding female or those found dead were related to the lack of maternal care, and were not due to an effect of the test item.

Histopathological findings:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

F1 Litter Responses, Litter Size, Sex Ratio and Survival Indices
Lower mean values were observed in the groups treated with the test item in litter size on day 1 (including and excluding dead fetuses this day), as well as on days 4 and 13 of lactation. The differences at 300 and 1000 mg/kg/day were significant in all cases, with the exception of days 4 (after cull) and 13.
There was no test-item-related effect on sex ratio, although statistically significant differences were observed at 300 and 1000 mg/kg/day in the total offspring, including those that died prior to the designated day 1.
Regarding offspring survival indices in the test-item-administered groups a dose-related decrease was observed in post-implantation and live birth indexes. No relevant differences were observed in the viability index on day 4 of lactation, but lactation index at 1000 mg/kg/day was considerably lower, compared to the Control group, with a 33% lower mean value.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

The number of live-born fetuses was reduced in a dose-related manner, as was the gestation index, in which the mean values of 300 and 1000 mg/kg/day were statistically significant.
There was no effect in offspring mean body weights. Regarding survival indices, there were lower mean values in post-implantation survival, live birth and viability indices when compared to Control, showing statistically significant differences at 300 and 1000 mg/kg/day.
In addition, at 1000 mg/kg/day, lactation index on day 13 was reduced with respect to Control values. Consequently, litter size in the test-item-administered groups was lower than that recorded in the Control group (mainly at 300 and 1000 mg/kg/day in which statistically significant differences were recorded). There were no offspring clinical or necropsy signs indicative of a reaction to Solvent Yellow 175, and there was no relevant effect on sex ratio or nipple areolae. Females at 300 and 1000 mg/kg/day showed significantly higher ano-genital distance mean values with respect to Control which were considered not relevant taking into account the Historical Control Data.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

not specified

Dose response relationship:

yes

F0 Cage signs – Pregnant Females – Mating – group distribution of observations

 

	Control	Solvent Yellow 175
Dose Group	1	2	3	4
Dose (mg/kg/day)	0	100	300	1000

 

Category	Observation	Day		Number of animals affected
			Group/Sex: Initial no:	1F 9	2F 9	3F 10	4F 10
Normal       Excreta	Within normal limits       Feces abnormal color, Dark         Urine abnormal color, Yellow	1 2 3   1 2 3 4 5   1 2 3 4 5		9 9 1   0 0 0 0 0   0 0 0 0 0	0 0 0   9 9 1 1 0   9 9 1 1 0	0 0 0   10 10 2 1 0   10 10 2 1 0	0 0 0   10 10 5 2 1   10 10 5 2 1

 

F0 Cage signs – Pregnant Females – Gestation – group distribution of observations

 

	Control	Solvent Yellow 175
Dose Group	1	2	3	4
Dose (mg/kg/day)	0	100	300	1000

 

Category	Observation	Day		Number of animals affected
			Group/Sex: Initial no:	1F 9	2F 9	3F 10	4F 10
Normal                                                 Excreta                                                       Excreta	Within normal limits                                                 Feces abnormal color, Dark                                                     Urine abnormal color, Yellow	0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23   0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26   0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26		9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 7 1   0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0   0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0	0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0   9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 8 6 2 0 0   9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 8 6 2 0 0	0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0   10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 9 4 0 0   10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 9 4 0 0	0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0   10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 9 4 1 1   10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 9 4 1 1

 

 

F0 Cage signs – Pregnant Females – Lactation – group distribution of observations

 

	Control	Solvent Yellow 175
Dose Group	1	2	3	4
Dose (mg/kg/day)	0	100	300	1000

 

Category	Observation	Day		Number of animals affected
			Group/Sex: Initial no:	1F 9	2F 9	3F 8	4F 7
Normal                                 Excreta                                 Excreta	Within normal limits                                 Feces abnormal color, Dark                               Urine abnormal color, Yellow	1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16   1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16   1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16		9 9 9 9 9 9 9 9 9 9 9 9 9 9 7 2   0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0   0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0	0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0   9 9 9 8 8 8 8 8 8 8 8 8 8 8 4 2   9 9 9 8 8 8 8 8 8 8 8 8 8 8 4 2	0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0   8 8 8 5 5 5 5 5 5 5 5 5 5 5 1 1   8 8 8 5 5 5 5 5 5 5 5 5 5 5 1 1	0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0   7 6 4 3 3 3 3 3 2 2 2 2 2 2 1 1   7 6 4 3 3 3 3 3 2 2 2 2 2 2 1 1

 

F0 Detailed signs – Pregnant Females –Mating – group distribution of observations

 

	Control	Solvent Yellow 175
Dose Group	1	2	3	4
Dose (mg/kg/day)	0	100	300	1000

 

Category	Observation	Day		Number of animals affected
			Group/Sex: Initial no:	1F 9	2F 9	3F 10	4F 10
Normal   Skin	Within normal limits   Encrustation, Buccal cavity	1   1		9   0	9   0	9   1	10   0

 

F0 Detailed signs – Pregnant Females – Gestation – group distribution of observations

 

	Control	Solvent Yellow 175
Dose Group	1	2	3	4
Dose (mg/kg/day)	0	100	300	1000

 

Category	Observation	Day		Number of animals affected
			Group/Sex: Initial no:	1F 9	2F 9	3F 10	4F 10
Normal                                 Behavior     Behavior                             Body Temperature   Breathing       Coat     Discharge       Eyelids   Muscle reaction   Posture     Skin color	Within normal limits                                 Decreased activity     Salivation                             Abnormally cold to touch   Irregular   Labored   Piloerection     Red, Anus   Red, Vaginal area   Partially closed, Bilateral   Abnormal gait, Uncoordinated   Hunched     Pallor, Whole body	0 6 7 8 9 10 12 13 14 15 16 17 18 19 20 26   22 23   0 6 8 9 10 12 13 14 15 16 17 18 19 20   22   23   22   22 23   22   23   23   22   22 23   22 23		9 0 9 0 0 0 0 0 9 0 0 0 0 0 9 0   0 0   0 0 0 0 0 0 0 0 0 0 0 0 0 0   0   0   0   0 0   0   0   0   0   0 0   0 0	9 0 9 0 0 0 0 0 9 0 0 0 2 0 9 0   0 0   0 0 0 0 0 0 0 0 0 0 0 2 0 1   0   0   0   0 0   0   0   0   0   0 0   0 0	10 0 10 0 1 0 0 2 10 3 6 2 1 2 10 0   0 1   0 0 0 1 0 0 2 1 3 6 2 1 2 2   0   0   0   0 1   0   1   1   0   0 1   0 1	10 1 10 2 1 1 2 5 10 4 4 4 4 4 10 1   1 1   1 1 2 1 1 2 5 5 4 4 4 4 4 3   1   1   1   1 1   1   1   1   1   1 1   1 1

 

F0 Detailed signs – Pregnant Females – Lactation – group distribution of observations

 

	Control	Solvent Yellow 175
Dose Group	1	2	3	4
Dose (mg/kg/day)	0	100	300	1000

 

Category	Observation	Day		Number of animals affected
			Group/Sex: Initial no:	1F 9	2F 9	3F 10	4F 10
Normal             Behavior	Within normal limits             Salivation	1 2 3 7 8 13   1		9 0 0 9 0 9   0	9 0 1 8 0 8   0	8 0 3 5 0 5   1	7 2 1 3 1 2   0

 

F0 Estrous cycles –group values before treatment

 

	Control	Solvent Yellow 175
Dose Group	1	2	3	4
Dose (mg/kg/day)	0	100	300	1000

 

Group /Sex	Number of animals		Regular 4 or 5 day cycles	Irregular cycle λ	Acyclic ψ
1F     2F     3F     4F	10     10     10     10	n (%)   n (%)   n (%)   n (%)	10 (100)   10 (100)   10 (100)   10 (100)	0     0     0     0	0     0     0     0

λ: At least one cycle of two, three or six to ten days        ψ: At least ten days without estrus

No test was applied as all animals had regular estrous cycles of 4 or 5 days.

 

F0 Estrous cycles –group values during treatment

 

	Control	Solvent Yellow 175
Dose Group	1	2	3	4
Dose (mg/kg/day)	0	100	300	1000

 

Group /Sex	Number of animals		Regular 4 or 5 day cycles	Irregular cycle λ	Acyclic ψ
1F     2F     3F     4F	10     10     10     10	n (%)   n (%)   n (%)   n (%)	10 (100)   9 (90)   10 (100)   8 (80)	0     1 (10)   0     2 (20)	0     0     0     0

λ: At least one cycle of two, three or six to ten days        ψ: At least ten days without estrus

The exact Linear-by-linear test with all groups included was not statistically significant (p=0.149)

 

F0 Pre-coital interval –group values

 

	Control	Solvent Yellow 175
Dose Group	1	2	3	4
Dose (mg/kg/day)	0	100	300	1000

 

Group /Sex	Number of animals		Pre-coital interval (days)
			1-4	5-8	9-12	13-14
1F     2F     3F     4F	10     10     10     10	n (%)   n (%)   n (%)   n (%)	10 (100)   10 (100)   10 (100)   10 (100)	-     -     -     -	-     -     -     -	-     -     -     -

Not test was applied as al animals have a pre-coital interval of between 1 and 4 days.

 

F0 Gestation length and gestation index –group values

 

	Control	Solvent Yellow 175
Dose Group	1	2	3	4
Dose (mg/kg/day)	0	100	300	1000

 

Group /Sex	Number of Pregnant animals		20	21	22	23	24	Number of live litters born	Gestation Index (%)
1F     2F     3F     4F	9     9     10     10	n (%)   n (%)   n (%)   n (%)	2 (22.2)   1 (11.1)   -     -	6 (66.)   2 (22.2)   -     -	1 (11.1)   4 (44.4)   4 (50)   3 (42.9)	-     1 (11.1)   4 (50)   4 (57.1)	0     1 (11.1)   -     -	9     8     5     2	100     89     50^     20^

For gestation length, the exact Linear-by-linear test with all groups included was statistically significant (p<0.01)

Upon exclusion of Group 4, the exact Linear-by-linear test was again statistically significant (p<0.01)

Upon exclusion of Group 3, the exact Linear-by-linear test was no longer statistically significant (p=0.062)

 

F0 Mating performance and fertility –group values

 

	Control	Solvent Yellow 175
Dose Group	1	2	3	4
Dose (mg/kg/day)	0	100	300	1000

 

Group /Sex	Number paired	Number mating	Number Achieving pregnancy	Percentage mating	Conception rate (%)	Fertility index (%)
Statistical test:				^1	CA	CA
1M 2M 3M 4M	10 10 10 10	10 10 10 10	9 9 10 10	100 100 100 100	90 90 100 100	90 90 100 100
Statistical test:				^1	CA	CA
1F 2F 3F 4F	10 10 10 10	10 10 10 10	9 9 10 10	100 100 100 100	90 90 100 100	90 90 100 100

¹No test was applied as there were no animals that did not mate.

 

F0 Stage of estrous cycle at termination –group values

 

	Control	Solvent Yellow 175
Dose Group	1	2	3	4
Dose (mg/kg/day)	0	100	300	1000

 

Group /Sex	Number smeared		Cycle stage at termination
			D	P
1F     2F     3F     4F	9     8     5     2	n (%)   n (%)   n (%)   n (%)	9 (100)   7 (88)   5 (100)   2 (100)	-     1 (12)   -     -

D: Diestrus

P: Pro-estrus

No test was applied as there were no animals in estrous

 

F1 Offspring survival indices –group mean values

 

	Control	Solvent Yellow 175
Dose Group	1	2	3	4
Dose (mg/kg/day)	0	100	300	1000

 

Group /Sex		Post implantation survival index (%)	Live birth index (%)	Viability index (%) Day 4	Lactation index (%) Day 13
Statistic test		Sh	CA	Fe
1F	Mean SD N N<100%	96.6 5.61 9 3	98.1 5.56 9 1	100.0 0.00 9 0	100.0 0.00 9 0
2F	Mean SD N N<100% % of 1F	90.3 10.57 9 6 94	79.5 40.89 9 2 81	100.0 0.00 8 0 100	100.0 0.00 8 0 100
3F	Mean SD N N<100% % of 1F	72.8* 29.91 9 8 75	47.4 44.08 9 7** 48	67.9 47.25 7 3 68	100.0 0.00 5 0 100
4F	Mean SD N N<100% % of 1F	57.8** 27.69 8 7 70	30.0 43.75 8 7** 70	100.0 0.00 3 0 100	66.7 57.74 3 1 67

The following data were used for the statistics tests, animal indices for post implantation survival index and animal indices dichotomized to 1 when 100% and 0 otherwise for live birth and viability indices.

 

F1 Litter size –group mean values

 

	Control	Solvent Yellow 175
Dose Group	1	2	3	4
Dose (mg/kg/day)	0	100	300	1000

 

Group /Sex		Implantations	Total @	Live on Day
				Before Cull		After Cull
				1	4	4	13
Statistics test		Wi	Wi	Wi	Wi
1F	Mean SD N	12.1 1.62 9	11.9 1.54 9	11.7 1.66 9	11.7 1.66 9	10.1 1.69 9	10.1 1.69 9
2F	Mean SD N % of 1F	11.6 3.13 9 95	10.7 3.04 9 90	8.8 3.69 8 75	8.8 3.69 8 75	7.8 2.92 8 77	7.8 2.92 8 77
3F	Mean SD N % of 1F	11.8 1.48 9 97	8.8* 3.96 9 74	5.0* 4.51 7 43	6.2* 4.32 5 53	5.4 3.36 5. 53	5.4 3.36 5 53
4F	Mean SD N % of 1F	12.0 1.51 8 99	8.3* 3.49 8 69	6.7* 4.93 3 57	6.7* 4.93 3 57	6.3 4.62 3 63	9.0 0.00 2 89

@ - Includes offspring that dies prior to the designated Day 1 of age

 

F1 Offspring sex ratio –group mean values

 

	Control	Solvent Yellow 175
Dose Group	1	2	3	4
Dose (mg/kg/day)	0	100	300	1000

 

Group /Sex		Total@			Live (before cull) on Day						Live (after cull) on Day
					1			4			4			13
		M	F	%M	M	F	%M	M	F	%M	M	F	%M	M	F	%M
Statistics test				Wi			Wi			Wi
1F	Mean SD N	7.4 2.01 9	4.4 2.46 9	63.6 19.66 9	7.3 2.06 9	4.3 2.45 9	63.8 19.62 9	7.3 2.06 9	4.3 2.45 9	63.8 19.62 9	7.3 2.06 9	2.8 1.86 9	72.8 17.40 9	7.3 2.06 9	2.8 1.86 9	72.8 17.40 9
2F	Mean SD N % of 1F	4.7 1.66 9 63	5.0 2.06 9 113	49.3 15.87 9 78	4.4 2.39 8 60	4.4 2.26 8 101	46.2 23.82 8 72	4.4 2.39 8 60	4.4 2.26 8 101	46.2 23.82 8 72	4.4 2.39 8 60	3.4 1.60 8 122	51.4 25.49 8 71	4.4 2.39 8 60	3.4 1.60 8 122	51.4 25.49 8 71
3F	Mean SD N % of 1F	3.2 1.86 9 43	4.1 2.71 9 93	42.2* 21.28 9 66	2.1 2.19 7 29	2.9 2.79 7 66	39.0 35.60 7 61	2.6 2.07 5 35	3.6 2.88 5 83	35.5 25.07 5 56	2.6 2.07 5 35	2.8 1.92 5 101	39.2 26.94 5 54	2.6 2.07 5 35	2.8 1.92 5 101	39.2 26.94 5 54
4F	Mean SD N % of 1F	2.9 2.30 8 39	3.4 2.07 8 76	42.3* 25.45 8 66	4.3 3.06 3 59	2.3 2.08 3 54	75.2 22.67 3 118	4.3 3.06 3 59	2.3 2.08 3 54	75.2 22.67 3 118	4.3 3.06 3 59	2.0 2.00 3 72	77.8 22.22 3 107	6.0 1.41 2 82	3.0 1.41 2 108	66.7 15.71 2 92

@ - Includes offspring that died prior to the designated Day 1 of age and excludes unsexed offspring

 

F1 Body weight –group mean values (g) for offspring

 

	Control	Solvent Yellow 175
Dose Group	1	2	3	4
Dose (mg/kg/day)	0	100	300	1000

 

Group /Sex		Day of age (before cull)		Day of age (after cull)
		1	4	4	7	13
Statistics test		Wi	Wi	Wi	Wi	Wi
1M	Mean SD N	5.89 0.736 9	8.77 1.394 9	8.77 1.394 9	14.47 1.860 9	28.27 2.862 9
2M	Mean SD N % of 1M	5.99 0.773 7 102	9.48 1.616 7 108	9.48 1.616 7 108	15.57 2.119 7 108	29.34 3.836 7 104
3M	Mean SD N % of 1M	5.41 0.499 5 92	8.86 0.932 4 101	8.86 0.932 4 101	14.62 1.258 4 101	27.94 0.878 4 99
4M	Mean SD N % of 1M	5.49 0.400 3 93	7.64 1.888 3 87	7.64 1.88 3 87	13.91 0.040 2 96	25.94 0.868 2 92
Statistics test		Wi	Wi	Wi	Wi
1F	Mean SD N	5.31 0.344 8	8.12 0.760 8	8.19 0.731 8	13.75 1.190 8	27.54 2.327 8
2F	Mean SD N % of 1F	5.62 0.721 8 106	9.01 1.648 8 111	8.94 1.700 8 109	15.14 2.112 8 110	28.78 3.584 8 105
3F	Mean SD N % of 1F	5.39 0.337 6 102	8.52 0.888 5 105	8.45 0.938 5 103	14.00 1.324 5 102	28.36 3.591 5 103
4F	Mean SD N % of 1F	5.40 0.573 2 102	8.31 0.441 2 102	8.04 0.815 2 98	13.10 1.473 2 95	25.16 1.559 2 91

 

F1 Ano-genital distance / Nipple/areolae –group mean litter values

 

	Control	Solvent Yellow 175
Dose Group	1	2	3	4
Dose (mg/kg/day)	0	100	300	1000

 

Group		MALES			FEMALES
		Body weight (g)	Ano-genital distance (mm)	Nipple/areolae	Body weight (g)	Ano-genital distance (mm)
		Day 1	Day 1	Day 13	Day 1	Day 1
Statistical test			Wi			Wi
1	Mean SD N	5.89 0.735 9	2.66 0.201 9	0 0 9	5.31 0.344 8	0.95 0.073 8
2	Mean SD N % of 1	5.99 0.773 7 102	2.67 0.268 7 100	0 0 7	5.62 0.721 8 106	1.04 0.142 8 109
2	Mean SD N % of 1	5.41 0.499 5 92	2.65 0.259 5 100	0 0 3	5.39 0.337 6 102	1.13* 0.160 6 118
4	Mean SD N % of 1	5.49 0.400 3 93	2.28 0.627 3 86	0 0 2	5.40 0.573 2 102	1.24* 0.249 2 130

For statistical analysis, ano-genital distance has been normalized using body weight as covariate.

Conclusions:

In conclusion, the effects of oral (gavage) administration of Solvent Yellow 175 to Wistar rats receiving 100, 300 or 1000 mg/kg/day for 14 days prior to mating and until sacrifice can be summarized as follows:
Reproductive / developmental toxicity:
- The No Observed Adverse Effect Level (NOAEL) for reproductive / developmental toxicity was considered to be 100 mg/kg/day, based on the increase in gestation length observed at 300 and 1000 mg/kg/day and the decrease in gestation index and viability indices when compared to Control.

Executive summary:

Introduction

The purpose of this study was to assess the general systemic toxic potential of Solvent Yellow 175 in rats, including a screen for reproductive/developmental effects and assessment of endocrine disruptor relevant endpoints, with administration of the test item by gavage for 5-8 weeks.

 

Three groups of 10 male and 10 female rats received Solvent Yellow 175 at the doses of 100, 300 and 1000 mg/kg/day. Males were treated continuously for two weeks before pairing up to necropsy, after a minimum of 36 consecutive days. Females were treated continuously for two weeks before pairing, throughout pairing and gestation, and until Day 13-15 of lactation (the day before sacrifice). Females were allowed to litter and rear their offspring, and litters were killed on Day 13-15 of lactation (the day before the corresponding female was killed).

F1 generation received no direct administration of the test item; any exposure was in utero or via the milk. A similarly constituted control group received the vehicle, corn oil.

 

During the study, mortality, clinical signs, sensory reactivity observations, grip strength, motor activity, body weight, food consumption, hematology, coagulation, blood biochemistry, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic examination were evaluated.

 

Clinical signs, behavior assessment, litter size, survival, sex ratio, body weight and macropathology were also assessed for all offspring.

 

Results

The study results can be summarized as follows:

- Preparation of all formulations was considered correct considering the acceptance ranges as formulation results demonstrated that all formulations were within the necessary acceptance range for the nominal concentration and coefficient of variation.

 

- Two females at both 300 and 1000 mg/kg/day were sacrificed for welfare reasons at the end of gestation period as they showed signs of difficulty during parturition.

Between late gestation and lactation day 8, one female at 100 mg/kg/day, three females at 300 mg/kg/day and six females at 1000 mg/kg/day were sacrificed due to total litter loss.

 

- Administration of Solvent Yellow 175 at the dose levels of 100, 300 or 1000 mg/kg/day had no other evident toxicological effects on clinical condition than those mentioned above, or in body weight, food consumption, motor activity, sensory reactivity and grip strength, pre-coital interval, estrous cycles, mating performance and fertility. A dose related increase in gestation length at increasing dose was observed, mainly due to the difficulties observed during parturition for some females. Dark feces and yellow urine recorded from day 1 of treatment onwards in all the test-item groups were attributable to the color of the test item. Salivation was recorded occasionally in test-item- administered males and females in all test-item groups as well as in a control male and female, probably due to taste aversion (from gavage dosing).

 

- Although they were not considered relevant and as a dose-response effect was not recorded, the hematocrit, hemoglobin and red blood cell values of the males sacrificed after 5 weeks of treatment with the test item showed significant differences, with mean values lower than those seen in the Control group. In addition, mean reticulocyte values higher than those of Controls were also found in males (statistically significant at 300 and 1000 mg/kg/day).

 

- There were no relevant differences considered related to the treatment in hematology in females or in coagulation and clinical biochemistry in males and females.

 

- T4 analyses of samples in F1 offspring on day 13 did not reveal any relevant differences that could be attributable to treatment. In males sacrificed at the end of treatment, statistically lower mean values were observed in the test-item-administered groups with respect to Control, although these differences were not dose-related.

 

- The macroscopic examination performed at the end of lactation period revealed changes in the stomach, cecum, thymus and kidneys of treated females.

 

- Histopathology revealed changes that were considered related to treatment with the test item in male thyroids and kidneys, female stomach and cecum, and thymus of both males and females. Changes observed in males, i.e. decrease in thymus weight and increase in thyroids and parathyroids, correlate with these findings. No treatment-related effects were detected in the reproductive organs or mammary glands.

 

- Regarding offspring survival indices, post implantation and live birth indexes decreased in a dose-related manner, and lactation index at 1000 mg/kg/day was lower with respect to Control.

No effect was observed in relation to the number of implantations but lower mean values were observed in the groups treated with the test item in litter size on days 1, 4 and 13 of lactation. The differences at 300 and 1000 mg/kg/day were significant in all cases.

In the offspring, there was no test-item-related effect of treatment on sex ratio, clinical signs, body weights or external macroscopic examination at sacrifice. Ano-genital distance showed a dose-related trend towards an increase at increasing dose in female pups.

 

Conclusion

In conclusion, the effects of oral (gavage) administration of Solvent Yellow 175 to Wistar rats receiving 100, 300 or 1000 mg/kg/day for 14 days prior to mating and until sacrifice can be summarized as follows:

 

Systemic toxicity:

− Considering that among the animals administered at 300 and 1000 mg/kg/day, two females in each dose were sacrificed for welfare reasons at late gestation (showing difficulties during parturition) and taking into account the histopathological findings observed in the digestive tract, thymus and thyroids and parathyroids, the No Observed Adverse Effect Level (NOAEL) could be established at 100 mg/kg/day.

 

Reproductive / developmental toxicity:

- The No Observed Adverse Effect Level (NOAEL) for reproductive / developmental toxicity was considered to be 100 mg/kg/day, based on the increase in gestation length observed at 300 and 1000 mg/kg/day and the decrease in gestation index and viability indices when compared to Control.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

K1

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 purpose of the study was to assess the general systemic toxic potential of Solvent Yellow 175 in rats, including a screen for reproductive/developmental effects and assessment of endocrine disruptor relevant endpoints, with administration of the test item by gavage for 5-8 weeks.

 

Three groups of 10 male and 10 female rats received Solvent Yellow 175 at the doses of 100, 300 and 1000 mg/kg/day. Males were treated continuously for two weeks before pairing up to necropsy, after a minimum of 36 consecutive days. Females were treated continuously for two weeks before pairing, throughout pairing and gestation, and until Day 13-15 of lactation (the day before sacrifice). Females were allowed to litter and rear their offspring, and litters were killed on Day 13-15 of lactation (the day before the corresponding female was killed).

F1 generation received no direct administration of the test item; any exposure was in utero or via the milk. A similarly constituted control group received the vehicle, corn oil.

 

During the study, mortality, clinical signs, sensory reactivity observations, grip strength, motor activity, body weight, food consumption, hematology, coagulation, blood biochemistry, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic examination were evaluated.

Clinical signs, behavior assessment, litter size, survival, sex ratio, body weight and macropathology were also assessed for all offspring.

 

Results

The study results can be summarized as follows:

- Two females at both 300 and 1000 mg/kg/day were sacrificed for welfare reasons at the end of gestation period as they showed signs of difficulty during parturition.

Between late gestation and lactation day 8, one female at 100 mg/kg/day, three females at 300 mg/kg/day and six females at 1000 mg/kg/day were sacrificed due to total litter loss.

- Administration of Solvent Yellow 175 at the dose levels of 100, 300 or 1000 mg/kg/day had no other evident toxicological effects on clinical condition than those mentioned above, or in body weight, food consumption, motor activity, sensory reactivity and grip strength, pre-coital interval, estrous cycles, mating performance and fertility. A dose related increase in gestation length at increasing dose was observed, mainly due to the difficulties observed during parturition for some females. Dark feces and yellow urine recorded from day 1 of treatment onwards in all the test-item groups were attributable to the color of the test item. Salivation was recorded occasionally in test-item- administered males and females in all test-item groups as well as in a control male and female, probably due to taste aversion (from gavage dosing).

- Although they were not considered relevant and as a dose-response effect was not recorded, the hematocrit, hemoglobin and red blood cell values of the males sacrificed after 5 weeks of treatment with the test item showed significant differences, with mean values lower than those seen in the Control group. In addition, mean reticulocyte values higher than those of Controls were also found in males (statistically significant at 300 and 1000 mg/kg/day).

- There were no relevant differences considered related to the treatment in hematology in females or in coagulation and clinical biochemistry in males and females.

- T4 analyses of samples in F1 offspring on day 13 did not reveal any relevant differences that could be attributable to treatment. In males sacrificed at the end of treatment, statistically lower mean values were observed in the test-item-administered groups with respect to Control, although these differences were not dose-related.

- The macroscopic examination performed at the end of lactation period revealed changes in the stomach, cecum, thymus and kidneys of treated females.

- Histopathology revealed changes that were considered related to treatment with the test item in male thyroids and kidneys, female stomach and cecum, and thymus of both males and females. Changes observed in males, i.e. decrease in thymus weight and increase in thyroids and parathyroids, correlate with these findings. No treatment-related effects were detected in the reproductive organs or mammary glands.

- Regarding offspring survival indices, post implantation and live birth indexes decreased in a dose-related manner, and lactation index at 1000 mg/kg/day was lower with respect to Control.

No effect was observed in relation to the number of implantations but lower mean values were observed in the groups treated with the test item in litter size on days 1, 4 and 13 of lactation. The differences at 300 and 1000 mg/kg/day were significant in all cases.

In the offspring, there was no test-item-related effect of treatment on sex ratio, clinical signs, body weights or external macroscopic examination at sacrifice. Ano-genital distance showed a dose-related trend towards an increase at increasing dose in female pups.

 

Conclusion

In conclusion, the effects of oral (gavage) administration of Solvent Yellow 175 to Wistar rats receiving 100, 300 or 1000 mg/kg/day for 14 days prior to mating and until sacrifice can be summarized as follows:

Systemic toxicity:

− Considering that among the animals administered at 300 and 1000 mg/kg/day, two females in each dose were sacrificed for welfare reasons at late gestation (showing difficulties during parturition) and taking into account the histopathological findings observed in the digestive tract, thymus and thyroids and parathyroids, the No Observed Adverse Effect Level (NOAEL) could be established at 100 mg/kg/day.

Reproductive / developmental toxicity:

- The No Observed Adverse Effect Level (NOAEL) for reproductive / developmental toxicity was considered to be 100 mg/kg/day, based on the increase in gestation length observed at 300 and 1000 mg/kg/day and the decrease in gestation index and viability indices when compared to Control.

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

In accordance with Regulation 1272/2008 (CLP), paragraph 3.7.2.2.1. classification as a reproductive toxicant is warranted when a substance has an intrinsic, specific property to produce adverse effects on reproduction and/or development without a non-specific secondary consequence of other toxic effects. From the OECD 422 study effects have been reported relating to decreased gestation index and viability indices when compared to the control animals at 300 and 1000 mg/kg/day, however these effects are not observed in absence of maternal toxicity. Therefore based on this information the substance cannot be adequately be classified for Reproductive Toxicity.