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REACH

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

EC number: 915-586-1 | CAS number: -

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

Administrative data

Description of key information

Repeated dose toxicity: oral - No Observed Adverse Effect Level (NOAEL) could be established at 100 mg/kg/day.

Key value for chemical safety assessment

Toxic effect type:: dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

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:

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

Details on route of administration:

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.

Vehicle:

corn oil

Details on oral 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.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical method Before commencement of treatment, an analytical method (M0384_HPLC_DFA_Solvent Yellow 175_Formulation_Vehicle_ISV) was validated in the present study. Formulations at two concentration levels (target concentrations: 1 and 200 mg/mL) were prepared and the following parameters were determined:
• System suitability (SST)
• Linearity
• Accuracy/repeatability
• Homogeneity
• Specificity
• Limit of Quantification (LOQ) and Limit of Detection (LOD)
• Stability [autosampler, formulations (20 ± 5 ºC, in the absence of light, for 8 days)]
This method also includes the procedure followed and the acceptance criteria for the analysis of administered formulations.
Analysis of the formulations administered: The formulations prepared at three different concentrations were analyzed twice over the course of the study to verify its correct 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.

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 the study.

Observations and examinations performed and frequency:

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 albumin 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)

Sacrifice and pathology:

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.

Other examinations:

Thyroid Hormone Analysis
Sequence of blood sampling on each occasion: In order to minimize any effect of the time of day on blood sampling, the samples were obtained within a comparable range of time according to the organization of the laboratory, thus allowing satisfactory comparisons between the groups.
Conditions: Following 6-8 hours of deprivation of food (except animals sacrificed for welfare reasons and those not confirmed to be not pregnant until necropsy, which were not fasted)
Anesthetic: Isoflurane
Sample site: retro-orbital sinus

Sample Identification
Primary tube
Kind of sample (blood); Study Number; Animal / Litter identification / Sex; Date of sampling; Day of extraction (At term /Lactation day 4 / Lactation day 13; Group of treatment; Aliquot number (1/1)
Secondary tube
Kind of sample (serum T4 / plasma TSH); Study Number; Animal / Litter identification / Sex; Date of sampling; Day of extraction (At term / Lactation day 4/ Lactation day 13; Group of treatment; Aliquot number (1/2 and 2/2)

Separation and storage: Centrifugation at 4 ºC for 10 minutes at 2000 g.
All available plasma/serum was transferred to appropriately labelled polypropylene cryotubes using plastic disposable pipettes.
The plasma/serum samples were stored at -80 ± 10 ºC, pending analysis.
Samples were sent in dry ice to the responsible scientist for their analysis.

Total number of samples: Terminal samples: 80 per parameter, 160 in total
Total: 114
Analysis: Samples from adult males were assessed for Thyroxine (T4) levels.
Sampling started on 22 January 2019 and analysis ended on 28 February 2019.

Method Thyroxine (T4)
Bioanalysis
Serum samples were analyzed at Envigo CRS, S.A.U. according to method
M0333_HPLC_BA_T4T3_Rat_Serum_Samples validated in Envigo GLP Study
SN83QL Thyroxine T4 / Triiodothyronine T3: Validation of an LC-MS/MS
Bioanalytical Method in Rat Serum and re-validated in Envigo GLP Study
NN19MX Thyroxine T4 / Triiodothyronine T3: Revalidation of an LC MS/MS
Bioanalytical Method in Rat Serum and cross validated in Envigo GLP Study
WF90VR 3,3,5’-Triiodo-L-Thyronine (T3) and Thyroxine (T4): Cross Validation of a Bioanalytical Method for the Determination of the Biomarker 3,3,5’-Triiodo-L-Thyronine (T3) and Thyroxine (T4) in Rat Serum using Liquid Chromatography with Tandem Mass Spectrometric Detection (LC-MS/MS).
GLP Study FF58YR (carried out at Envigo CRS Limited, UK) investigates the stability of samples for a storage period of up to one year. To date, T4 stability has been proven for 387 days at -70 ± 10 ºC.

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)

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:

no effects observed

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

Description (incidence and severity):

In males, adjusted epididymides, liver and thyroids and parathyroids weights were higher than in Controls. These differences were not dose-related and were within/close to that observed in the historical control range.
A dose-related trend towards a decrease in adjusted thymus weight was observed. Significant differences were observed at 300 and 1000 mg/kg/day.
Adjusted uterus, cervix and oviducts weight in females was higher than in Controls in all test item-administered groups (100, 300 and 1000 mg/kg/day), attaining statistical significance with respect to Control at 300 and 1000 mg/kg/day. Differences were dose-related.
At 1000 mg/kg/day, mean adjusted brain weight was found to be statistically higher than that recorded in the Control group. However, based on the fact that the mean value corresponds to a sample size of only two females in the group, it cannot be considered relevant.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The macroscopic examination performed 5-8 weeks after treatment revealed the following changes in the stomach, cecum, thymus and kidneys of treated females.
Stomach: A small dark area in the nonglandular mucosa was noted in a female receiving 1000 mg/kg/day.
Cecum: A small cecum was seen in a female receiving 1000 mg/kg/day.
Thymus: A small thymus was seen in some females receiving 1000 mg/kg/day. Only one female given 100 mg/kg/day showed a similar change.
Kidneys: Pale kidneys were seen in a female given 1000 mg/kg/day.
Yellow coloration of the adipose tissue and/or gastric mucosa in treated animals (all male dose groups and high dose group females) were attributed to the test item color.
All the other gross findings were considered to be incidental and unrelated to the test item. A small spleen was noted in a female receiving 1000 mg/kg/day. However, it was not available for histological examination.

Neuropathological findings:

not examined

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.

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

mortality

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

F0 Body weight – Males – group mean values (g)

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

Group /Sex

Day

Statistics test

Mean

260.7

4.93

287.7

8.49

315.3

11.62

334.5

15.74

351.6

6.65

366.0

16.78

382.4

18.13

391.8

15.96

Mean

% of 1M

262.9

9.36

101

290.4

9.46

101

319.0

14.48

101

334.6

15.84

100

350.4

20.39

100

364.1

22.34

377.9

26.57

386.7

27.96

Mean

% if 1M

261.8

13.22

100

286.9

14.42

100

317.5

18.88

101

333.3

22.87

100

351.6

23.31

100

363.8

24.91

382.1

26.85

100

391.3

28.85

100

Mean

% of 1M

261.8

9.56

100

290.3

11.95

101

317.9

14.47

101

337.2

17.83

101

356.2

20.21

101

370.0

22.57

101

387.1

27.40

101

398.7

29.50

102

F0 Body weight – Pregnant Females – group mean values (g)

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

Group /Sex

Day

Statistics test

Mean

184.2

5.05

197.8

6.63

210.0

8.45

216.9

10.33

226.6

10.97

225.3

10.97

250.4

14.04

284.4

14.35

337.5

17.28

251.3

19.18

266.9

17.11

298.9

11.67

Mean

% of 1F

187.1

6.85

102

202.5

7.61

102

214.1

7.90

102

217.1

6.31

100

227.0

6.94

100

231.1

6.40

103

257.9

5.51

103

283.1

6.87

100

329.5

11.77

247.2

17.40

268.5

10.05

101

287.3

12.78

Mean

% if 1F

185.0

8.20

100

198.5

7.08

100

211.6

8.63

101

217.6

8.48

100

228.8

9.22

101

229.0

9.84

102

253.4

1.47

101

279.6

9.19

326.8

12.97

256.8

12.68

102

270.4

6.70

101

288.5

6.74

Mean

% of 1F

184.9

7.24

100

196.6

9.02

211.8

10.62

101

219.2

11.13

101

229.9

8.53

101

231.4

8.97

103

256.4

10.73

102

279.4

14.73

326.5

10.23

237.4

24.10

271.6

12.96

102

286.9

2.53

F0 Cage signs – Males – 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:

Normal

Excreta

Within normal limits

Feces abnormal color, Dark

Urine abnormal color, Yellow

P10

P11

P12

P13

P14

P15

F0 Cage signs – Pregnant Females – Pre-test/Treatment – 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:

Normal

Excreta

Within normal limits

Feces abnormal color, Dark

Urine abnormal color, White

Urine abnormal color, Yellow

P10

P11

P12

P13

P14

P15

F0 Detailed signs – Males – 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:

Normal

Behavior

Build (Deformity)

Coat

Skin

Skin color

Within normal limits

Salivation

Swollen area, Forelimb digit(s)

Hair loss, Dorsal surface

Encrustation, Upper dorsal surface

Abnormal color, Blue, Forelimb – right

F0 Detailed signs – Pregnant Females – Pre-Test/Treatment - 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:

Normal

Behavior

Breathing

Skin

Within normal limits

Salivation

Labored

Encrustation, Buccal cavity

FOB: Sensory activity – group mean values

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

Group /Sex	Estimuli	Pinna reflex (1-3)	Blink reflex	Pupil closure reflex	Proprioception (right leg) (1-3)	Tail pinch response (1-4)	Auditory Startle reflex (1-4)	Righting reflex (1-4)
	Grade	2	Both eye pass	Both eye pass	1	3	3	1
1M	No. of animals	5	5	5	5	5	5	5
2M	No. of animals	5	5	5	5	5	5	5
3M	No. of animals	5	5	5	5	5	5	5
4M	No. of animals	5	5	5	5	5	5	5
1F	No. of animals	5	5	5	5	5	5	5
2F	No. of animals	5	5	5	5	5	5	5
3F	No. of animals	5	5	5	5	5	5	5
4F	No. of animals	2	2	2	2	2	2	2

FOB: Grip Strength – group mean values (g)

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

Group /Sex

Grip strength

Forelimb 1 (g)

Forelimb 2 (g)

Forelimb 3 (g)

Forelimb Mean (g)

Hindlimb 1 (g)

Hindlimb 2 (g)

Hindlimb 3 (g)

Hindlimb Mean (g)

Statistical test

Mean

607.30

108.022

70.12

342.716

903.48

300.741

760.30

212.162

728.94

269.227

612.64

178.493

745.58

283.905

695.72

212.186

Mean

% of 1M

897.18

386.111

148

1260.28

278.776

164

842.84

390.873

1000.10

205.105

132

617.78

144.105

609.50

138.034

601.06

215.249

609.45

76.243

Mean

% of 1M

770.84

258.477

127

648.84

133.218

848.68

349.208

756.12

149.906

869.30

343.094

119

918.96

247.299

150

822.66

259.808

110

870.31

177.572

125

Mean

% of 1M

812.70

431.720

134

814.00

292.471

106

887.30

270.406

838.00

215.373

110

745.84

260.695

102

667.06

210.799

109

692.5

130.550

701.79

165.624

101

Mean

405.46

101.983

504.56

126.230

460.28

113.901

456.77

83.930

492.42

137.485

381.44

57.296

391.30

95.192

421.72

89.226

Mean

% of 1F

611.94

183.364

151

584.72

186.129

116

524.24

124.122

114

573.63

128.028

126

675.56

78.452

137

520.02

165.705

136

603.80

126.418

154

599.79**

67.315

142

Mean

% of 1F

732.38

144.501

181

591.20

145.061

117

477.78

53.726

104

600.45

74.231

131

707.20

186.008

144

658.54

153.719

173

596.02

137.910

152

653.92**

80.454

155

Mean

% of 1F

498.10

109.036

123

646.25

164.827

128

410.20

9.051

518.18

94.304

113

672.70

101.965

137

705.55

192.404

185

653.65

63.145

167

677.30**

119.171

161

FOB: Motor activity – group mean values (beam counts)

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

Group /Sex

Motor activity

10 (min)

20 (min)

30 (min)

40 (min)

50 (min)

60 (min)

Total (min)

Statistical test

Mean

380.6

136.21

219.8

152.06

114.8

104.57

169.4

133.62

122.6

138.02

165.6

141.67

1172.8

699.69

Mean

% of 1M

463.6

103.02

122

253.8

133.90

115

135.4

88.02

118

199.4

149.18

118

132.6

98.05

108

55.4

54.43

1240.2

359.40

106

Mean

% of 1M

543.6

137.25

143

327.0

178.69

149

138.2

80.50

120

125.4

89.21

175.8

85.15

143

110.2

135.67

1420.2

236.97

121

Mean

% of 1M

448.2

175.81

118

215.2

83.28

184.0

91.44

160

146.8

128.64

102.8

140.37

226.0

252.44

136

1323.0

477.98

113

Mean

132.0

56.87

302.6

122.48

198.2

128.05

159.2

85.72

175.8

70.38

181.6

55.30

1149.4

104.94

Mean

% of 1F

200.8*

41.55

152

169.8

93.86

179.4

27.12

132.6

61.80

220.6

194.49

125

165.4

80.55

1068.6

323.12

Mean

% of 1F

317.8**

52.83

241

207.12

67.78

130.4

86.83

192.0

125.49

121

217.0

51.57

123

209.8

144.22

116

1274.2

323.68

111

Mean

% of 1F

293.0**

12.73

222

259.0

89.10

110.5

51.62

263.0

31.11

165

213.0

57.98

121

159.5

60.10

1298.0

21.21

113

F0 Hematology and Coagulation – group values

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

Group /Sex

Hct

L/L

g/dL

RBC x10¹²/L

Retic x10¹²/L

MCH

MCHC

g/dL

MCV

WBC x10⁹/L

x10⁹/L

Statistical test

Mean

0.457

0.0160

15.1

0.59

8.79

0.194

0.117

0.0192

17.2

0.74

33.1

0.48

52.1

1.85

5.31

2.266

0.90

0.266

4.22

2.008

Mean

0.419**

0.0240

14.0*

0.69

8.00*

0.570

0.144

0.0244

17.6

0.71

33.4

0.31

52.5

1.91

6.82

1.122

1.21

0.360

5.45

1.216

Mean

0.422**

0.0181

14.2*

0.67

8.18*

0.229

0.156*

0.0302

17.4

0.51

33.7

0.21

51.5

1.27

5.60

3.497

1.06

0.282

4.40

3.426

Mean

0.422**

0.0131

14.2*

0.38

8.29*

0.336

0.174**

0.0204

17.1

0.83

33.6

0.49

51.0

2.30

7.77

1.303

1.11

0.326

6.47

1.174

Group /Sex

x10⁹/L

LUC

x10⁹/L

Plt

x10⁹/L

SPT

Secs

SAPT

Secs

Statistical test

Mean

0.05

0.021

0.00

0.005

0.09

0.042

0.03

0.015

749

37.4

16.9

0.76

18.8

2.80

Mean

0.03**

0.004

0.00

0.004

0.09

0.030

0.04

0.015

749

98.6

16.8

1.04

18.7

4.48

Mean

0.02**

0.009

0.00

0.004

0.09

0.036

0.03

0.034

774

61.8

16.2

0.87

18.6

3.00

Mean

0.02**

0.011

0.01

0.005

0.11

0.047

0.06

0.011

800

82.6

16.8

0.43

19.7

1.25

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

Group /Sex

Hct

L/L

g/dL

RBC x10¹²/L

Retic x10¹²/L

MCH

MCHC

g/dL

MCV

WBC x10⁹/L

x10⁹/L

Statistical test

Mean

0.440

0.0191

14.6

0.47

7.86

0.419

0.209

0.0506

18.5

0.82

33.1

0.97

56.0

1.48

5.97

1.512

2.81

1.288

2.99

0.714

Mean

0.409

0.0196

13.9

1.07

7.50

0.429

0.180

0.0377

18.6

1.02

34.0

1.57

5.95

1.067

5.95

1.067

2.46

0.297

3.21

0.813

Mean

0.437

0.0106

14.7

0.32

7.76

0.167

0.132

0.0573

18.9

0.70

33.6

0.17

56.4

2.11

5.57

1.436

1.77

0.954

3.54

0.772

Mean

0.433

0.0099

14.1

1.63

7.87

0.078

0.184

0.0926

17.9

1.84

32.5

2.90

55.0

0.71

4.31

3.288

1.65

4.61

Group /Sex

x10⁹/L

LUC

x10⁹/L

Plt

x10⁹/L

SPT

Secs

SAPT

Secs

Statistical test

Mean

0.04

0.010

0.00

0.006

0.17

0.047

0.05

0.030

817

241.8

16.2

0.67

24.6

1.84

Mean

0.03

0.015

0.01

0.005

0.20

0.106

0.05

0.019

859

198.3

15.9

0.51

25.3

5.34

Mean

0.03

0.017

0.00

0.005

0.18

0.083

0.05

0.012

786

156.1

15.5

0.30

21.3

2.65

Mean

0.02

0.01

0.25

0.10

612

116.7

16.7

0.42

31.3

9.19

F0 Clinical Biochemistry – group values

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

Group /Sex

ALP

U/L

ALT

U/L

AST

U/L

Bi Ac μmol/L

Urea mmol/L

Creat μmol/L

Gluc mmol/L

Chol mmol/L

HDL mmol/L

Statistical test

Mean

138

32.6

12.3

12.0

199

138.3

37.1

19.10

4.10

0.974

2.6

9.23

0.651

2.06

0.388

1.67

0.279

Mean

115

26.6

15.8

15.1

301

108.3

18.2

12.69

4.56

0.499

31*

4.0

8.78

2.028

1.77

0.198

1.33

0.116

Mean

121

35.5

9.7

6.7

195

34.2

21.5

15.61

4.16

0.388

2.2

7.94

1.003

2.24

0.201

1.76

0.154

Mean

115

16.4

54.6

28.1

299

243.0

29.5

6.08

4.30

0.318

1.5

9.10

0.504

2.30

0.310

1.873

0.172

Group /Sex

LDL mmol/L

Trig mmol/L

Na mmol/L

mmol/L

Ca mmol/L

Phos mmol/L

Total Prot g/L

Alb

g/L

Statistical test

Mean

0.34

0.124

0.60

0.054

141

0.5

4.0

0.20

0.9

2.56

0.071

1.93

0.173

1.1

0.8

Mean

0.35

0.067

0.43

0.181

143

0.9

3.8

0.30

100

0.2

2.47

0.053

1.91

0.115

2.0

0.8

Mean

0.40

0.093

0.51

0.249

142

1.7

4.1

0.24

100

1.9

2.50

0.052

1.94

0.166

2.3

1.4

0.5

Mean

0.41

0.180

0.61

0.302

140*

0.9

4.3

0.27

1.0

2.53

0.043

1.90

0.064

2.3

0.9

10*

0.7

Group /Sex

g/L

Beta

g/L

Gamma g/L

A/G

Ratio

ALB%

A1G%

A2G%

BGL%

GGL%

Statistical test

Mean

0.2

0.1

0.4

1.15

0.039

53.4

0.87

18.4

1.08

8.9

0.31

17.4

0.38

1.9

0.70

Mean

0.2

0.8

0.4

1.11

0.053

52.5

1.19

17.4

1.34

9.6

0.41

18.2*

0.73

2.2

0.59

Mean

0.4

0.6

0.3

1.13

0.060

53.0

1.30

17.6

0.73

9.3

0.56

18.6**

0.77

1.5

0.38

Mean

0.4

12**

0.4

1.11

0.044

52.6

0.98

16.8*

0.77

9.6

0.61

19.4**

0.15

1.5

0.54

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

Group /Sex

ALP

U/L

ALT

U/L

AST

U/L

Bi Ac μmol/L

Urea mmol/L

Creat μmol/L

Gluc mmol/L

Chol mmol/L

HDL mmol/L

Statistical test

Mean

101

48.1

21.5

15.8

208

61.2

47.6

46.53

7.48

1.712

3.1

8.34

1.213

2.52

0.384

1.91

0.318

Mean

15.6

16.3

102

38.5

199

60.5

40.3

29.48

7.21

1.491

4.6

7.96

0.990

2.79

0.313

1.96

0.394

Mean

37.8

18.9

28.3

181

61.0

61.4

64.18

7.04

1.936

7.2

7.88

1.695

2.45

0.313

1.96

0.283

Mean

125

5.6

8.1

140*

41.9

378**

59.8

104.3

79.97

7.93

0.856

4.2

6.91

0.608

2.43

0.134

1.83

0.078

Group /Sex

LDL mmol/L

Trig mmol/L

Na mmol/L

mmol/L

Ca mmol/L

Phos mmol/L

Total Prot g/L

Alb

g/L

Statistical test

Mean

0.24

0.085

1.75

0.805

136

1.3

4.0

0.53

2.0

2.59

0.113

2.71

0.455

1.9

1.7

0.7

Mean

0.33

0.096

1.58

0.688

135

3.3

4.2

0.55

3.2

2.59

0.211

2.67

0.470

7.2

3.5

2.0

Mean

0.24

0.094

1.04

0.376

135

4.4

3.8

0.36

3.2

2.53

0.116

2.11

0.94

3.1

2.1

0.5

Mean

0.20

0.014

1.83

1.358

134

0.0

4.4

0.25

0.2

2.65

0.106

2.87

0.134

4.5

1.2

1.5

Group /Sex

g/L

Beta

g/L

Gamma g/L

A/G

Ratio

ALB%

A1G%

A2G%

BGL%

GGL%

Statistical test

Mean

0.6

0.7

0.3

1.09

0.099

52.2

2.22

17.6

1.19

10.5

0.98

17.5

1.09

2.2

0.48

Mean

0.6

1.3

0.3

1.08

0.067

51.8

1.60

16.0

1.59

10.9

0.63

19.1

0.95

2.1

0.28

Mean

0.4

0.6

1.27

0.070

55.9

1.38

14.3**

0.78

9.6

0.95

18.2

0.69

2.1

0.90

Mean

1.2

2.6

0.4

1.24

0.316

54.9

6.36

13.7**

1.48

10.5

1.27

19.3

3.04

1.7

0.57

F0 Macropathology – Males – group distribution of findings

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

Number of animals affected

Tissue/Organ and Findings

Group/Sex

No. of animals

Number of animals within normal limits

Adipose Tissue

Abnormal color

Cecum

Abnormal color

Kidneys

Dilated Pelvis

Lymph Node, Mandibular

Abnormal color

Lymph Node, Mesenteric

Abnormal color

Skin and Subcutis

Deformity

Scab(s)

Stomach

Abnormal color

Thymus

Abnormal color

[CA]

[Ch]

[CA]

[Ch]

7**

6**

10**

Statistic test indicated in brackets; a dash indicates statistics were not possible and a space indicates statistics were not required.

F0 Macropathology – Pregnant Females – group distribution of findings

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

Number of animals affected

Tissue/Organ and Findings

Group/Sex

No. of animals

Number of animals within normal limits

Adipose Tissue

Abnormal color

Adrenals

Enlarged

Cecum

Small

Ileum

Abnormal color

Kidneys

Abnormal color

Dilated Pelvis

Liver

Nodule

Lymph Node, Mandibular

Enlarged

Lymph Node, Mesenteric

Abnormal color

Spleen

Small

Stomach

Abnormal color

Dark area(s)

Thymus

Small

[CA]

[Ch]

[CA]

5**

Statistic test indicated in brackets; a dash indicates statistics were not possible and a space indicates statistics were not required.

F0 Organ Weights – Males – group mean absolute and adjusted values (g)

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

Group /Sex

Terminal Body weight

Adrenals

Brain

Prostate, Seminal Vesicles and Coagulating Gland

Epididymides

Heart

Kidneys

Liver

Spleen

Testes

Thymus

Thyroids and Parathyroids

Statistics test

Mean

385.89

15.74

0.072

0.010

2.048

0.097

2.656

0.740

1.335

0.374

0.996

0.032

2.203

0.136

10.840

0.771

0.703

0.098

3.585

0.707

0.468

0.153

0.024

0.004

Mean

% of 1M

382.61

25.25

0.077

0.013

108

2.11

0.067

103

2.923

0.488

110

1.449

0.210

109

1.018

0.099

102

2.307

0.197

105

11.732

0.693

108

0.782

0.062

111

3.818

0.221

106

0.382

0.117

0.032

0.004

131

Mean

% of 1M

387.48

30.69

100

0.077

0.005

106

2.114

0.089

103

2.896

0.240

109

1.432

0.202

107

1.083

0.117

109

2.413

0.084

110

13.995

1.961

129

0.714

0.132

102

3.855

0.279

108

0.296

0.045

0.027

0.003

112

Mean

% of 1M

393.30

23.97

102

0.071

0.007

2.094

0.045

102

2.662

0.488

100

1.593

0.145

119

1.054

0.049

106

2.341

0.072

106

13.806

1.090

127

0.785

0.124

112

3.943

0.187

110

0.259

0.033

0.007

135

Statistics test

Adjusted Mean

0.072

0.078

0.076

0.072

2.050

2.103

2.126

2.088

2.664

2.949

2.895

2.629

1.336

1.452

1.432

1.589*

0.994

1.027

1.070

1.060

2.201

2.314

2.403

2.345

10.830

11.775

13.933**

13.836**

0.702

0.786

0.708

0.788

0.467

0.387

0.290*

0.262**

0.024

0.032*

0.027*

0.032**

F0 Organ Weights – Pregnant Females – group mean absolute and adjusted values (g)

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

Group /Sex

Terminal Body weight

Adrenals

Brain

Heart

Kidneys

Liver

Ovaries

Spleen

Thymus

Thyroids and Parathyroids

Uterus and Cervix and Oviducts

Statistics test

Mean

284.48

19.99

0.100

0.016

1.950

0.094

0.918

0.039

1.682

0.094

12.289

0.746

0.093

0.012

0.706

0.051

0.237

0.053

0.027

0.004

0.624

0.073

Mean

% of 1F

269.87

20.10

0.089

0.008

1.910

0.068

0.834

0.042

1.676

0.058

100

11.815

0.890

0.101

0.020

109

0.662

0.146

0.198

0.026

0.007

0.710

0.199

114

Mean

% of 1F

271.16

11.94

0.101

0.025

101

1.966

0.076

101

0.883

0.070

1.758

0.086

105

12.425

1.963

101

0.101

0.023

108

0.677

0.144

0.237

0.081

100

0.028

0.008

105

1.006**

0.346

161

Mean

% of 1F

250.54**

39.07

0.102

0.009

101

2.042

0.109

105

0.948

0.089

103

1.707

0.033

101

12.937

1.234

105

0.109

0.021

117

0.673

0.038

0.158

0.034

0.027

0.008

101

1.773**

1.233

284

Statistics test

Adjusted Mean

0.100

0.090

0.101

0.102

1.931

1.920

1.966

2.067*

0.908

0.839

0.883

0.961

1.681

1.677

1.758

1.708

12.398

11.761

12.427

12.794

0.095

0.101

0101

0.106

0.703

0.663

0.677

0.228

0.203

0.237

0.171

0.025

0.026

0.028

0.029

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:
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.

Executive summary:

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).

During the study, mortality, clinical signs, sensory reactivity observations, grip strength, motor activity, body weight, food consumption, hematology, coagulation, blood biochemistry, organ weight and macroscopic examination were evaluated.

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. 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 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.

Conclusion

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.

Endpoint conclusion

Endpoint conclusion:: adverse effect observed
Dose descriptor:: NOAEL
: 100 mg/kg bw/day
Study duration:: subacute
Species:: rat
Quality of whole database:: K1
System:: gastrointestinal tract
Organ:: oesophagus; parathyroid gland; stomach; thymus; thyroid gland

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:: no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:: no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:: no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:: no study available

Additional information

Repeated dose toxicity: oral

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.

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

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

Conclusion

Systemic toxicity:

Justification for classification or non-classification

In accordance with Regulation 1272/2008 (CLP), classification guidance values specified under Table 9.3.9 (10 < C ≤ 100 mg/kg bw/day) and taking into consideration Harber's rule specified under paragraph 3.9.2.9.5 for differences in study duration, the substance is classified as STOT RE 2 on the basis of NOAEL 100 mg/kg/day and effects noted on vital organs: oesophagus, stomach, thymus, thyroid gland and parathyroid gland.

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