1,4-bis[(2-ethyl-6-methylphenyl)amino]anthraquinone

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

There was no fertility study on Reinblau RLW.

 

Key information on Solvent Green 28 (Category member): NOAEL for systemic toxicity and reproductive / developmental toxicity = 1000 mg/kg bw/day (OECD 422, GLP, K, Rel.1)

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Justification for type of information:

Justification Document for the Category of Six Anthraquinone Dyes

LANXESS Deutschland GmbH has registered five mono-constituent anthraquinone dyes of similar chemical structure using a category approach: Solvent Violet 36 (CAS No 82-16-6), Solvent Green 3 (CAS No 128-80-3), Reinblau RLW (CAS No 41611-76-1), Reinblau BLW (CAS No 32724-62-2) and Solvent Green 28 (CAS No 4851-50-7). Additional data were taken from another registered anthraquinone dye, Solvent Blue 104 (CAS No 116-75-6), leading to a category consisting of six members (see attached justification in the Category dossier).

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no signs were observed in association with dose administration.
Clinical signs observed at routine examination comprised several incidences of vocalisation and irritable behaviour in males and females at all dose levels, including Controls. One female receiving 1000 mg/kg/day was recorded to have a twisted muzzle on Day 14 of lactation. No test item-related changes in general clinical condition were observed following treatment with Macrolex Grün G.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no premature deaths.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

For males receiving Macrolex Grün G, overall mean body weight gains between Weeks 0-5 were marginally low at all dose levels when compared with Control values. This was primarily due to a slight dose dependent reduction in body weight gain observed in males between Weeks 0-1, following commencement of treatment, with statistical significance observed at 1000 mg/kg/day.
During the two-week pre-pairing treatment period, females receiving 100 mg/kg/day were unaffected by administration with Macrolex Grün G. For females receiving 300 mg/kg/day, a reduction in body weight gain was observed between Weeks 0-1 (50% lower than Control). Females given Macrolex Grün G at 1000 mg/kg/day exhibited a similar decrease in body weight gain between Weeks 1-2. Mean body weight gain was unaffected when the whole premating period (Weeks 0-2) was considered.
During the gestation period for females receiving Macrolex Grün G at doses up to and including 1000 mg/kg/day, a dose dependent reduction in overall body weight gains was observed (Days 0-20), a lthough no statistical significance was observed.
Following parturition, body weight performance was similar to that of Controls for females receiving 100, 300 and 1000 mg/kg/day up to Day 13 of lactation.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption for males receiving Macrolex Grün G at doses up to and including 1000 mg/kg/day was considered to be unaffected by treatment.
For females receiving Macrolex Grün G at doses of 100, 300 and 1000 mg/kg/day, food consumption values were similar to those of the Controls throughout the treatment period, gestation and lactation phases.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not specified

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The hematological examination of peripheral blood performed after five weeks of treatment for males and on Day 14 of lactation for females did not reveal any toxicologically significant differences from control.
All inter-group differences, including those attaining statistical significance, were minor, confined to one sex or lacked dose-relationship and were therefore attributed to normal biological variation.
Such differences included the slightly low neutrophil and eosinophil concentrations in males receiving 100, 300 or 1000 mg/kg/day, low prothrombin times in males at 100, 300 and 1000 mg/kg/day, high platelet count in males at 1000 mg/kg/day and high reticulocyte count in females at 100, 300 and 1000 mg/kg/day.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The biochemical examination of plasma performed after five weeks of treatment for males and on Day 14 of lactation for females did not reveal any toxicologically significant differences from control.
All inter-group differences, including those attaining statistical significance, were minor, confined to one sex or lacked dose-relationship and were therefore attributed to normal biological variation. Such differences included the slightly, but statistically significantly low alkaline phosphatase in males at 300 and 1000 mg/kg/day and alanine aminotransferase in males at 100, 300 and 1000 mg/kg/day and the slightly, but statistically significantly high cholesterol levels in females at 1000 mg/kg/day. Low creatinine concentration was evident in males at 1000 mg/kg/day.

Endocrine findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not specified

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Treatment Related Findings
There were no treatment related changes.

Incidental Findings
Mineralisation was seen in the kidneys (cortex, sometimes correlated with macroscopically pale kidneys) and the glandular mucosa of the stomach of control and treated females. In the stomach; minimal mineralisation was seen in controls and treated females at a similar incidence and was considered unrelated to treatment. In the kidneys; histopathological findings associated with the observed distribution of mineralisation included minimal to slight degeneration/necrosis and minimal dilatation of cortical tubules. The incidence and/or severity of the kidney mineralisation and associated findings appeared slightly increased in treated females compared with controls.
A compilation of background control data (BCD) from similar recent studies was performed for the all above findings. In this BCD, mineralisation of the kidney cortex and cortical tubular degeneration/necrosis each have an incidence range of 0 – 100% and a severity range of minimal to moderate. Cortical tubular dilatation has a BCD incidence range of 0 – 83.3%, with a severity range of minimal to
moderate. Mineralisation of the glandular stomach mucosa has a BCD incidence range of 0 – 60%, with a severity range of minimal to moderate. All these findings within the current study are well within these ranges.
Therefore, the discussed findings are considered to be incidental, with the apparent slight increase in treated females due to chance and not related to treatment.
The incidence and distribution of all other findings were considered to be unrelated to treatment.
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.

Histopathological findings: neoplastic:

not specified

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Thyroid Hormone Analysis
There was no effect of treatment on the circulating levels of thyroxine (T4) in adult males.

Sensory Reactivity Observations and Grip Strength
The sensory reactivity observations conducted during Week 5 of treatment for males and Day 7-9 of lactation for females revealed no findings which were considered treatment related in animals receiving Macrolex Grün G at doses of 100, 300 and 1000 mg/kg/day.

Motor Activity
The motor activity assessment conducted during Week 5 of treatment revealed no test-item related effects in male or female animals receiving Macrolex Grün G at all dose levels. It was noted that in male animals receiving 1000 mg/kg/day, a lower than Control statistical significance was seen in the low beam break at the 6 minute testing point, as well as a higher than Control statistical significance seen in the low beam break at 60 minutes. These inconsistent, isolated findings were considered not to be associated with administration of Macrolex Grün G.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

All females allocated to study showed normal 4/5 day estrous cycles during the acclimatization period.

All females showed diestrus at termination.

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

Estrous cyclicity, pre-coital interval, gestation length, mating performance, fertility and gestation index were considered unaffected by treatment with Macrolex Grün G.

Key result

Dose descriptor:

NOAEL

Remarks:

(systemic toxicity)

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

haematology

clinical biochemistry

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

Key result

Dose descriptor:

NOAEL

Remarks:

(Reproductive toxicity)

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

No signs were recorded that were considered to be related to parental treatment with Macrolex Grün G.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

All females that mated were pregnant.
Litter size, offspring survival to Day 13 of age and sex ratio were unaffected by parental treatment with Macrolex Grün G.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The mean body weight performance of both male and female offspring up to Day 13 of age remained similar to those of the Controls at all dose levels. Thus, there were no treatment-related effects on body weights of offspring of parental animals given Macrolex Grün G at doses of 100, 300 or 1000 mg/kg/day.

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 specified

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

The macroscopic examination of offspring revealed no test item related lesions.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Offspring Ano-genital Distance
Mean ano-genital distances in males and females were considered to be unaffected by treatment.

Thyroid Hormone Analysis
There was considered to be no effect of treatment upon T4 levels in offspring on Day 13 of age.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

mortality

clinical biochemistry

gross pathology

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Thyroid Hormone Analysis

There was no effect of treatment on the circulating levels of thyroxine (T4) in adult males or in offspring on Day 13 of age.

Group	Treatment	Dose		Adult Male at Termination (pg/mL)	Day 13 of age Offspring (pg/mL)
		(mg/kg/day)			Male	Female
1	Corn oil	0	Mean	28500	29700	33400
			SD	9340	6580	13400
			CV	32.8	22.2	40.1
			N	10	10	10
2	Macrolex Grün G	100	Mean	27600	37700	37000
			SD	11000	7420	5300
			CV	39.9	19.7	14.3
			N	10	10	10
3	Macrolex Grün G	300	Mean	26700	36000	38500
			SD	12500	6540	7660
			CV	46.8	18.2	19.9
			N	10	10	10
4	Macrolex Grün G	1000	Mean	32000	28800	37400
			SD	8570	5390	7890
			CV	26.8	18.7	21.1
			N	10	10	10

 

Summary of findings in the gastrointestinal tract for animals killed after 5 weeks of treatment

Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Dose (mg/kg/day)	0	100	300	500	0	100	300	500
Finding	Abnormal Contents, Green
Caecum	0	3	6	9	0	1	1	4
Colon	0	3	5	9	0	0	1	2
Ileum	0	0	1	2	0	0	1	1
Jejunum	0	0	2	3	0	0	0	0
Rectum	0	0	3	6	0	0	0	2
Stomach	0	1	4	4	0	2	2	6
Number of tissues examined	10	10	10	10	10	10	10	10

Summary of findings in the stomach for animals killed after 5 weeks of treatment

Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Dose (mg/kg/day)	0	100	300	1000	0	100	300	1000
Abnormal colour, Green	0	2	5	9	0	8	7	9
Number of tissues examined	10	10	10	10	10	10	10	10

Summary of general comments for animals killed after 5 weeks of treatment

Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Dose (mg/kg/day)	0	100	300	1000	0	100	300	1000
Fur stained, Green	0	0	0	8	0	1	0	0
Tail stained, Green	0	0	0	5	0	1	2	6
Number of tissues examined	10	10	10	10	10	10	10	10

Conclusions:

It was concluded that the oral administration of Macrolex Grün G to parental Sprague-dawley rats at dose levels of 100, 300 or 1000 mg/kg/day administered for two weeks before pairing, during pairing and then up to termination of the males after five weeks of treatment and females on Day 14 of lactation, was well tolerated.
Reproductive performance, fertility, litter size and offspring survival and growth were unaffected by parental treatment and, in the context of this study, Macrolex Grün G showed no evidence of being an endocrine disruptor.
The no-observed adverse-effect level (NOAEL) of Macrolex Grün G for systemic toxicity was considered to be 1000 mg/kg/day and the no-observed adverse-effect level (NOAEL) of Macrolex Grün G for reproductive/developmental toxicity was also considered to be 1000 mg/kg/day.

Executive summary:

Summary

The purpose of this study was to assess the potential systemic toxic potential in Crl:CD(SD) rats, including a screen for reproductive/developmental effects and assessment of endocrine disruptor relevant endpoints, with administration of the test item, Macrolex Grün G, by oral administration forat least five weeks.

Three groups of ten male and ten female rats receivedMacrolex Grün Gat doses of 100, 300 or 1000 mg/kg/day by oral gavage administration. Males were treated daily for two weeks before pairing, up to necropsy after a minimum of five consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 13 of lactation. Females were allowed to litter, rear their offspring and were killed on Day 14 of lactation. The 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, at the same volume dose as the treated groups.

During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, hematology (peripheral blood), blood chemistry, thyroid hormone analysis, estrous cycles, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic pathology and histopathology investigations were undertaken.

The clinical condition, litter size and survival, sex ratio, body weight, ano-genital distance and macropathology for all offspring were also assessed. Nipple counts were performed on male offspring on Day 13 of age. 

Results

F0 responses

Administration with Macrolex Grün G at doses up to and including 1000 mg/kg/day was considered to generally be well tolerated throughout the treatment period. There were no premature deaths, no dosing signs and no clinical signs observed in association with dose administration.

For males receiving Macrolex Grün G, overall mean body weight gains between Weeks 0-5 were marginally low at all dose levels when compared with Control values. This was primarily due to a slight dose dependent reduction in body weight gain observed in males between Weeks 0-1, following commencement of treatment, with statistical significance observed at 1000 mg/kg/day.

During the two-week pre-pairing treatment period, females receiving 100 mg/kg/day were unaffected by administration withMacrolex Grün G. For females receiving 300 mg/kg/day, a reduction in body weight gain was observed between Weeks 0-1 (50% lower than Control). Females given Macrolex Grün G at 1000 mg/kg/day exhibited a similar decrease in body weight gain between Weeks 1-2.Mean body weight gain wasunaffectedwhen the whole premating period (Weeks 0-2) was considered.During the gestation period for females receiving Macrolex Grün G at doses up to and including 1000 mg/kg/day, a dose dependent reduction in overall body weight gains was observed (Days 0-20), although no statistical significance was observed. Following parturition, body weight performance was similar to that of Controls for females receiving 100, 300 and 1000 mg/kg/day up to Day 13 of lactation.

Estrous cyclicity, pre-coital interval, gestation length, mating performance, fertility and gestation index were unaffected by treatment.

The hematological examination of blood and the biochemical examination of plasma did not reveal any conclusive effect of treatment and there was no effect upon circulating levels of thyroxine (T4) in adult males.

Macroscopic findings were limited to green content, representing the colour of the test item, seen in the caecum, colon, ileum, jejunum, rectum and stomach in treated animals with a dosedependent increase in incidence and greencolour of the stomach seen in treated animals with a dose dependent increase in incidence.Histopathological findings in the full range of tissues examined were considered to be unrelated to treatment. Organ weights were considered unaffected by treatment.

F1 responses

The clinical condition, litter size, sex ratio, survival indices and body weight and weight gain of offspring were unaffected by parental treatment.

There was no effect of parental treatment upon circulating levels of thyroxine (T4) in offspring on Day 13 of age.

The ano-gential distances of offspring were unaffected by paternal treatment and no nipples were seen on any male offspring on Day 13 of age.

No macroscopic findings considered to be related to paternal treatment were recorded.

 

Conclusion

It was concluded that the oral administration of Macrolex Grün G to parental Sprague-dawley rats at dose levels of 100, 300 or 1000 mg/kg/day administeredfor two weeks before pairing, during pairing and then up to termination of the males after five weeks of treatment and females on Day 14 of lactation, was well tolerated.

Reproductive performance, fertility, litter size and offspring survival and growth were unaffected by parental treatment and,in the context of this study,Macrolex Grün Gshowed no evidence of being an endocrine disruptor.

The no-observed adverse-effect level (NOAEL) of Macrolex Grün G for systemic toxicity was considered to be 1000 mg/kg/day and the no-observed adverse-effect level (NOAEL) of Macrolex Grün G for reproductive/developmental toxicity was also considered to be
1000 mg/kg/day.

Reference 2

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the extended one-generation reproductive toxicity study does not need to be conducted because there are no results from available repeated dose toxicity studies that indicate adverse effects on reproductive organs or tissues, or reveal other concerns in relation with reproductive toxicity

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING

According to REACH Regulation (EC) No 1907/2006 Annex IX, 8.7.3 Column 2, the extended one generation study shall be proposed by the registrant "if the available repeated dose toxicity studies (e.g. 28-day or 90-day studies, OECD 421 or 422 screening studies) indicate adverse effects on reproductive organs or tissues or reveal other concerns in relation with reproductive toxicity." The existing 28-day and OECD 414 studies did not rise any alerts on reproductive toxicity. Therefore, testing in a one-generation study is not justified at this tonnage level (REACH Registration 100 -1000 tonnes).

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The study on Solvent green 28 - one of the category members - is GLP-compliant and of high quality Klimisch score =1)

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

There was no fertility study with Reinblau RLW. No effects on reproductive organs were observed in a 28 day study in rats. The pathologic evaluation consisted of organ weight, gross and microscopic examination of reproductive organs, incl. tetes and ovaries. No treatment-related changes were observed for any reproductive organ investigated during macroscopic and microscopic examination of all major organs (NOAEL, rat: 1000 mg/kg bw/day; males and females).

 

The following studies are available for category members:

 

1/ Solvent Green 28 (CAS 4851-50-7): In an OECD guideline 422 study (Combined Repeated Dose Toxicity Study and Reproductive/ Developmental Toxicity Screening Study in the Crl:CD(SD) Rat by Oral Gavage Administration) Solvent Green 28 was administered to three groups of ten male and ten female rats by oral gavage administration, for approximately 6 weeks (males) and up to eight weeks (females) (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 100, 300 and 1000 mg/kg bw/day. A similarly constituted control group received the vehicle, corn oil, at the same volume dose as the treated groups.

The no-observed adverse-effect level (NOAEL) of Solvent Green 28 for systemic toxicity was considered to be 1000 mg/kg bw/day and the no-observed adverse-effect level (NOAEL) of Solvent green 28 for reproductive/developmental toxicity was also considered to be 1000 mg/kg bw/day.

 

2/ Solvent Blue 104 (Source : ECHA disseminated dossier) : An OECD test guideline (OECD 421) and GLP-compliant Reproduction/Developmental Toxicity Screening Test was performed with the test item (Solvent Blue 104). Groups of 10 male and 10 female Wistar rats received doses of 0, 100, 300 and 1000 mg/kg bw by daily gavage. No toxicologically significant changes were noted in any of the parameters investigated in this study.

An NOAEL for general toxicity of 1000 mg/kg/day was established. No test item-related effects were observed in males or females up to and including 1000 mg/kg bw/day, the highest dose level administered.
An NOAEL for reproduction and development of 1000 mg/kg/day was established. No test item-related effects on reproduction or development were observed up to and including 1000 mg/kg bw/day, the highest dose level administered.

 

 Conclusion:

The available two screening tests for reproductive toxicity and development toxicity consistently show no toxicity of the anthraquinone dye group; up to the limit dose no adverse effects are reported, and it can be concluded that “no hazard” is identified in any of these studies.

Effects on developmental toxicity

Description of key information

Developmental toxicity: via oral route: NOAEL = 1000 mg/kg bw/day (OECD 414, GLP, K, Rel. 1)

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 4 December 2015 to 29 January 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study performed according to the OECD TG 414 (2001 version) without deviation.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

2001

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

1998

Deviations:

no

Principles of method if other than guideline:

One group of 22 females received Macrolex Blau 3R at the limit dose of 1000 mg/kg bw/day by daily oral gavage administration at a dose volume of 10 mL/kg bw/day from Day 6 to 19 after mating. A similarly constituted control group received the vehicle, 0.5% CMC-Na solution, according to the same dose volume and regimen. Animals were killed on Day 20 after mating for reproductive assessment and fetal examination.
Clinical observations, body weight and food consumption were recorded. Adult females were examined macroscopically at necropsy on Day 20 after mating with the weight of the gravid uterus recorded. All fetuses were weighed, subjected to an external macroscopic examination and then subsequently to detailed internal visceral or skeletal examination.

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at ambient temperature in the dark
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: The homogeneity and stability of formulations during storage were confirmed as part of a separate study (Sponsor Study No. 7L447). In that GLP-compliant study, the stability and homogeneity of the test material in the vehicle at concentrations of 0.4 and 100 mg/mL were confirmed as eight days under refrigerated storage conditions
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: n/a
- Reactivity of the test material with the incubation material used (e.g. plastic ware): n/a

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): The required amount of test substance was ground in a mortar using a pestle and mixed with some vehicle to form apaste. Furtheramountsofvehicleweregraduallyadded and mixed to produce a smooth, pourable suspension. The suspension was quantitatively transferred and diluted to volume and finally mixed using a high-shear homogeniser.
- Preliminary purification step (if any): not applicable

FORM AS APPLIED IN THE TEST (if different from that of starting material) not applicable

Species:

rat

Strain:

other: Crl:CD(SD)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited
- Age at study initiation: approximately 70 days of age
- Weight at study initiation: 232 to 279 g
- Fasting period before study: none
- Housing: up to four animals during acclimatisation; individually during gestation. Solid (polycarbonate) bottom cages, containing softwood based bark-are fibre bedding which was changes at appropriate intervals each week.
- Diet (e.g. ad libitum): SDS VRF1 Certified pellet diet ad libitum. The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
- Water (e.g. ad libitum): potable water from the public supply ad libitum.
- Acclimation period: 5 days before commencement of dosing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 40-70%
- Air changes (per hr): n/a (filtered fresh air which was passed to atmosphere and not recirculated)
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 9 December 2015 From: To: 8 January 2015

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5% CMC-Na solution

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The required amount of test substance was ground in a mortar using a pestle and mixed with some vehicle to form apaste. Furtheramountsofvehicleweregraduallyadded and mixed to produce a smooth, pourable suspension. The suspension was quantitatively transferred and diluted to volume and finally mixed using a high-shear homogeniser.

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly

VEHICLE
- Justification for use and choice of vehicle (if other than water): the same vehicle was used for the 28-day study
- Concentration in vehicle: 100 mg/mL
- Amount of vehicle (if gavage): volume dose = 10 mL/kg bw
- Lot/batch no. (if required): n/a
- Purity: n/a

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The samples were analysed in accordance with the validated Envigo Analytical Procedure FIA/M118/15.
The analytical method involved dispersion in water (5 mL) followed by dissolution in acetonitrile. Extracted samples were diluted in acetonitrile/water 70/30 v/v followed by reverse phase high performance liquid chromatographic analysis with ultra violet detection at 254 nm. Sample concentrations were determined with reference to external standards prepared in the concentration range 1 |ig/mL to 5 µg/mL.
For Week 1 and the Last week oftreatment, freshly prepared test formulations were sampled (4 x 1 mL, 2 x 3 mL for controls, accurately weighed) by Pharmacy personnel and submitted for analysis. Duplicate samples were analysed in accordance with the analytical procedure, and the samples were retained for contingency. These were discarded following acceptable results.
The mean concentrations of Macrolex Blau 3R in test formulations analysed for the study were within 1% of nominal concentrations, confirming accurate formulation. The precision of individual results from the mean value was within 3%, confirming the precision of analysis. Procedural recovery results confirmed the continued accuracy of the method and were not used to correct the final reported values.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Verification of same strain and source of both sexes: yes, a colony of stud males was maintained specifically for the purpose of mating; these animals were not part of the study and were maintained as stock animals.
- Proof of pregnancy: Ejected copulation plugs in cage tray and vaginal smears were checked for the presence of sperm referred to as day 0 of pregnancy. Only females showing at least two copulation plugs were allocated.
- Any other deviations from standard protocol:

Duration of treatment / exposure:

Females were treated from Day 6 to Day 19 (inclusive) after mating, once daily at approximately the same time each day.

Frequency of treatment:

Once daily

Duration of test:

21 days

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

22 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels for this main embryo-fetal study in the rat were based on the results of a preliminary embryo-fetal rat toxicity study performed in Sprague Dawley rats (Envigo Study No. TMR0140). In that study there were no toxicologically relevant observed effects at 1000 mg/kg bw/day upon the dams or on embryo-fetal survival or growth. Based upon this absence of effects in the preliminary study, and the known low solubility and very low bioavailability of the test item, it was considered that the limit test design was appropriate for this test item, investigating a single high dose level of 1000 mg/kg bw/day.
- Rationale for animal assignment (if not random): To group and cage position in the sequence of mating. Females mating on any one day were evenly distributed amongst the groups.
Allocation was controlled to prevent any stock male from providing more than one mated female in each treatment group.
- Fasting period before blood sampling for (rat) dam thyroid hormones: not applicable
- Time of day for (rat) dam blood sampling: not applicable
- Other: n/a

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: n Days 0, 5, 12, 18 and 20 after mating

BODY WEIGHT: Yes
- Time schedule for examinations: on Days 0, 3, and 6-20 after mating

FOOD CONSUMPTION : Yes
The weight of food supplied to each adult, that remaining and an estimate of any spilled was recorded for the periods Days 0-2, 3-5, 6-9, 10-13, 14-17 and 18-19 after mating inclusive.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on Day 20 after mating
- Organs examined: All adult animals were subject to a detailed necropsy. Afterareviewofthehistoryofeach animal, a full macroscopic examination of the tissues was performed. All external features andorificeswereexaminedvisually. Anyabnormalityintheappearanceorsizeofanyorgan and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative. Particular attention was paid to organ colour, taking into account that the test material is blue, with appropriate data recording.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes (including cervix and ovaries):
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Blood sampling:

- Plasma: No
- Serum: No

Fetal examinations:

- External examinations: Yes: (all per litter)
- Soft tissue examinations: Yes: [ half per litter]
- Skeletal examinations: Yes: [ half per litter]
- Head examinations: Yes: [ half per litter]
- Anogenital distance of all live rodent pups: no

Statistics:

The following data types were analysed at each timepoint separately:
Body weight, using absolute values and gains over appropriate study periods Gravid uterine weight and adjusted body weight
Food consumption, over appropriate study periods
Litter size and survival indices
Fetal, placental and litter weight
(further details in the field "Any other information materials and methods incl. tables")

Indices:

Pre-implantation loss (%) = (Number of corpora lutea - Number of implantations) / Number of corpora lutea x 100.
Post-implantation loss (%) = (Number of implantations - Number of live fetuses) / Number of implantations x 100.

Historical control data:

Control Incidence Major / Minor Skeletal / Minor Visceral abnormalities - Crl:CD(SD)Rat - from 8 studies are included. (Cf. attachement to the ESR)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

At 1000 mg/kg bw/day a total of 17 females were recorded with the clinical sign of blue staining of the tail. This was considered to be related to the blue coloured test material being excreted in the faeces. Two animals were recorded with abnormally coloured staining of the head and muzzle, where a further qualifier of colour was omitted in error, however these were recorded on Day 0 after mating, and therefore could not have been related to the test material. In addition four treated females (numbers 31,32, 33 and 34) were recorded with abnormal colouration of the tail on Day 20 after mating; however, no further qualifier of colour was recorded in error. Three out of the four of these females were confirmed as having a blue tail at necropsy on Day 20 after mating.
There were no other consistent clinical signs, nor any dosing signs, observed that were considered to be related to treatment at the limit dose of 1000 mg/kg bw/day.

Mortality:

no mortality observed

Description (incidence):

There were no premature decedents.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean overall bodyweight gain during gestation was similar to controls and unaffected by treatment at 1000 mg/kg/day.
There were a number of incidences of slight but statistically significant differences in mean absolute body weight at 1000 mg/kg/day when compared to the Controls (approximately 3% on Days 6, 10, 11, 12, 16, 17, 18 of gestation), however, this was principally due to slightly lower gain experienced during Days 3 to 6 of gestation, prior to the commencement of treatment, such that mean bodyweight at the start of treatment was statistically significantly lower than Controls for females receiving 1000 mg/kg/day. After the commencement of treatment, mean body weight gain during Days 6-20 of gestation for treated females was not significantly different from Controls (Controls 113g; dose group 111g).

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Mean overall values of food consumption during Days 6-19 of gestation for females receiving 1000 mg/kg bw/day were no different from Controls and were unaffected by treatment.
Mean food consumption for treated females immediately prior to the commencement of treatment (during Days 3-5 of gestation) was marginally lower than Controls, attaining statistical significance, however, subsequent food consumption for the remainder of gestation was unaffected by treatment.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Mean gravid uterine weight was similar to Controls and unaffected by treatment and when mean values of body weight and body weight gain were adjusted for the contribution of the gravid uterus, the maternal portion of mean body weight gain during Days 6-20 of gestation was similar to Controls and unaffected by treatment at 1000 mg/kg bw/day.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Several females receiving 1000 mg/kg bw/day showed blue colouration of gastro-intestinal content.
Three females at 1000 mg/kg bw/day showed blue colouration of the rectal tissue, 5 females showed blue coloration of the stomach tissue and 17 females were confirmed as having blue colouration of the tail.
The blue staining observed was considered discolouration caused by the test material itself (a blue dye) and is considered not to represent toxicity.
There were no other blue coloured organs observed in any female, and no further maternal findings observed at macroscopic examination that were considered to relate to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Number of abortions:

no effects observed

Description (incidence and severity):

One Control female was found to be non-pregnant. All treated females were pregnant with a live litter on Day 20 of gestation.

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Details on maternal toxic effects:

Embryo-fetal survival was clearly unaffected by treatment at 1000 mg/kg bw/day with mean numbers of implantations, resorptions, live young and percentages of sex ratio and pre and post-implantation loss being similar to Control values.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

changes in number of pregnant

changes in pregnancy duration

clinical biochemistry

clinical signs

dead fetuses

early or late resorptions

effects on pregnancy duration

gross pathology

haematology

histopathology: non-neoplastic

maternal abnormalities

mortality

necropsy findings

number of abortions

organ weights and organ / body weight ratios

pre and post implantation loss

total litter losses by resorption

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean male, female and overall fetal weights in litters of females receiving 1000 mg/kg bw/day were lower than Controls, and although statistical significance was attained, the difference was marginal (approximately 0.2g or 5-6% lighter than Controls) and considered not adverse since embryo-fetal survival and development were clearly unaffected by treatment.
Mean placental weights at 1000 mg/kg/day were similar to Controls and unaffected by treatment.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

Fetal development was clearly unaffected by treatment at the limit dose of 1000 mg/kg bwday.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The incidence of major and minor abnormalities and skeletal variants showed no relationship to treatment.
At 1000 mg/kg bw/day there was a slightly increased incidence of delayed/ incomplete ossification/unossified thoracic vertebrae compared to concurrent control, however, this was within the Historical Control range.

Visceral malformations:

no effects observed

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

changes in sex ratio

fetal/pup body weight changes

changes in litter size and weights

changes in postnatal survival

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

Based on the results of this embryo-fetal developmental toxicity study in rats, it was concluded that the No-Observed--Adverse-Effect-Level (NOAEL) of Macrolex Blau 3R for maternal toxicity was the limit dose of 1000 mg/kg bw/day.
For embryo-fetal development, the limit dose of 1000 mg/kg bw/day was considered to be the No-Observed-Adverse-Effect-Level (NOAEL), when administered during the organogenesis and fetal growth phases of gestation in the rat, as there was a marginal reduction in fetal weight which was considered not adverse due to there being no effect of treatment upon embryo-fetal survival or development.

Executive summary:

The objective of this study was to assess the influence of Macrolex Blau 3R, a blue powder, on embryo-fetal survival and development when administered during the organogenesis and fetal growth phases of pregnancy in the Sprague Dawley CD rat. The study was conducted according to the OECD TG 414 and in compliance with GLP.

One group of 22 females received Macrolex Blau 3R suspended in 0.5% CMC-Na solution at the limit dose of 1000 mg/kg bw/day by daily oral gavage administration at a dose volume of 10 mL/kg/day from Day 6 to 19 after mating. A similarly constituted Control group received the vehicle, according to the same dose volume and regimen. Animals were killed on Day 20 after mating for reproductive assessment and fetal examination.

Clinical observations, body weight and food consumption were recorded. Adult females were examined macroscopically at necropsy on Day 20 after mating with the weight of the gravid uterus recorded. All fetuses were weighed, subjected to an external macroscopic examination and then subsequently to detailed internal visceral or skeletal examination.

 

Treatment of pregnant female Sprague Dawley rats with Macrolex Blau 3R daily from Day 6 to Day 19 after mating, inclusive, at the limit dose of 1000 mg/kg bw/day was well tolerated and elicited no deaths and no toxicity related changes in clinical condition of the adult females.

At 1000 mg/kg bw/day a total of 17 females were recorded with the clinical sign of blue staining of the tail. At necropsy several females receiving 1000 mg/kg/day showed blue colouration of gastro-intestinal contents, three females showed blue colouration of rectal tissue, 5 females were recorded with blue coloration of stomach tissue and 17 females were confirmed as having blue colouration of the tail. The blue staining observed was considered discolouration caused by the test material itself (a blue dye) and is considered not to represent toxicity.

Body weight gain and food consumption during gestation were unaffected by treatment and there were no other maternal findings observed at macroscopic examination that were considered to relate to treatment.

There was no effect of treatment at 1000 mg/kg bw/day upon embryo-fetal survival or placental weights, and fetal development was also unaffected by treatment at this limit dose, with the incidence of major and minor abnormalities and skeletal variants showing no relationship to treatment.

Mean male, female and overall fetal weights at 1000 mg/kg bw/day were lower than Controls, but the difference was considered marginal (approx. 5-6%) and was clearly not adverse since embryo fetal survival and development were unaffected by treatment.

 

Based on the results of this embryo-fetal developmental toxicity study in rats, it was concluded that the No-Observed-Adverse- Effect-Level (NOAEL) of Macrolex Blau 3R for maternal toxicity and embryo-fetal development was the limit dose of 1000 mg/kg bw/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The available study is GLP-compliant and of high quality (Klimisch score = 1)

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

A developmental toxicity study according to OECD TG 414 was conducted in rats. Reinblau RLW was administered during the organogenesis and fetal growth phases of pregnancy in the Sprague Dawley CD rat. One group of 22 females received Reinblau RLW suspended in 0.5% CMC-Na solution at the limit dose of 1000 mg/kg bw/day by daily oral gavage administration at a dose volume of 10 mL/kg bw/day from Day 6 to 19 after mating. A similarly constituted Control group received the vehicle alone. Animals were killed on Day 20 after mating for reproductive assessment and fetal examination. Clinical observations, body weight and food consumption were recorded. Adult females were examined macroscopically at necropsy on Day 20 after mating with the weight of the gravid uterus recorded. All fetuses were weighed, subjected to an external macroscopic examination and then subsequently to detailed internal visceral or skeletal examination.

 

Based on the results of this embryo-fetal developmental toxicity study in rats, it was concluded that the No-Observed-Adverse- Effect-Level (NOAEL) of Reinblau RLW for maternal toxicity and embryo-fetal development was the limit dose of 1000 mg/kg bw/day.

Toxicity to reproduction: other studies

Additional information

no data

Justification for classification or non-classification

Harmonised classification:

The substance has no harmonised classification according to the Regulation (EC) No. 1272/2008 (CLP). 

 

Self-classification:

Based on the reproductive and developmental toxicity studies performed on Reinblau RLW or on Category members a non-classification for fertility and development is justified.