Registration Dossier

Administrative data

Description of key information

Groups of Sprague Dawley rats, comprising 5 animals of each sex, were treated at dose levels of 0 and 1000 mg/kg/day. 
All animals treated with 1000 mg/kg/day of the analogous substance, Brown HH 469, survived the 4 week study period. No treatment-related changes in clinical condition or behaviour occurred. Similar overall body weight gains were recorded in both test and control groups. Food consumption was similar in both groups. There were no changes in the haematological and clinical chemistry parameters measured. All organ weights and organ/body weight ratios were considered similar for treated and control animals. The gross and histological appearance of the skin site treated was similar to that of control animals. There was no evidence of any systemic toxicity in organs examined at necropsy or in tissues selected for histological evaluation.
In conclusion, dermal application of the analogous substance at a dose level of 1000 mg/kg/day produced no evidence of systemic toxicity.

Key value for chemical safety assessment

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:
2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: recent guideline study according to GLP, screening study for reprductive effects
Qualifier:
according to
Guideline:
other: OECD 421
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
- Animals: Rat, RccHanTM: WIST(SPF)
- Rationale: Recognized by international guidelines as a recommended test system.
- Breeder: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
- Number of Animals: 44 males (11 per group) and 44 females (11 per group)
- Age (at Start of Treatment): 11 weeks
- Body Weight Range (at Start of Treatment): 295 to 329 g (males) and 178 to 213 g (females)
- Identification: Cage card and individual animal number (ear tattoo).Pups were individually tattooed with Indian ink on day 1 post partum.
- Randomization: Performed after at least three days of acclimatization using a computer-generated random algorithm. Body weights (recorded on the day of allocation) were taken into consideration in order to ensure similar mean body weights in all groups.
- Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS

- Conditions: Standard laboratory conditions. Air-conditioned with 10 - 15 air changes per hour, continuously monitored environmental conditions (temp. range: 22 ± 3 °C; relative humidity range: 30 - 70%). Values for both temperature and humidity were outside these ranges but were transient variations and were considered not to have any influence on the study and, therefore, these data are not reported but retained at Harlan Laboratories. There was 12 hour fluorescent light / 12-hour dark cycle with music during the light period.
- Accommodation: In groups of five in Makrolon type-4 cages with wire mesh tops up to the day of randomization and afterwards individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’ J. Rettenmaier & Söhne GmbH & CoKG, 73494 Rosenberg / Germany, imported by Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) with paper enrichment (ISO-BLOX from Harlan Laboratories B.V., Netherlands) (batch/lot nos. 02105120601 and 6960C.CS-100099). During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles.
- Diet: Pelleted standard Harlan Teklad 2018C (batch no. 80/11) rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum. The feed batch was analyzed for contaminants.
- Water: Community tap-water from Füllinsdorf was available ad libitum in water bottles. A bacteriological assay, chemical and contaminant analyses of representative samples were performed.
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
DOSE FORMULATIONS

The dose formulations were prepared weekly using the test item as supplied by the Sponsor.

Acid Brown 425 was weighed into a glass beaker on a tared precision balance and approximately 80% of the vehicle was added (w/v). Using a magnetic stirrer, a homogeneous suspension was prepared. Having obtained a homogeneous mixture, the remaining vehicle was added. Separate formulations were prepared for each concentration. Dose formulations were divided into daily aliquots.

Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

STORAGE OF DOSE FORMULATIONS

Dose formulations were stored in refrigerator (5 ± 3 °C) in brown bottles in daily aliquots.

Based upon the results of stability analyses performed within the Harlan Laboratories non-GLP study D51147 (Dose Range-Finding Study for a Reproduction/Developmental Toxicity Screen¬ing Test in the Han Wistar Rat), dose formulations were stable for at least one week in refrigerator.

TREATMENT

- Method: Oral, by gavage
- Rationale for Method: Administration by gavage is a common and accepted route of exposure for this type of studies.
- Frequency of Administration: Once daily
- Target Dose Levels: 0 mg/kg bw/day (group 1, control group), 100 mg/kg bw/day (group 2), 300 mg/kg bw/day (group 3) and 1000 mg/kg bw/day (group 4)
- Rationale for Dose Level Selection: The dose levels were selected based on a previous non-GLP dose range-finding toxicity study in Han Wistar rats, Harlan Laboratories study D51147, using dose levels of 0, 100, 300 and 1000 mg/kg/ day. No adverse toxic effects were found at any dose level.
- Dose Volume: 10 mL/kg body weight
- Dose Concentrations: 0 mg/mL (group 1), 10 mg/mL (group 2), 30 mg/mL (group 3) and 100 mg/mL (group 4)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
METHOD

On the first treatment day samples from the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples of about 2 g of each concentration were taken from the middle to confirm the stability (4 hrs at room temperature and 8 days in refrigerator). During the last week of the treatment, samples were taken from the middle to confirm concentration. The aliquots for analysis of dose formulations were frozen (-20 ± 5 °C) and delivered on dry ice to the responsible person for formulation analysis (Harlan Laboratories Ltd., Zelgliweg 1, 4452 Itingen / Switzerland) and stored there at -20 ± 5 °C until analysis.

The samples were analyzed by UV-VIS following an analytical procedure developed at Harlan Laboratories study D51160. The test item was used as the analytical standard. Analyzed samples were not discarded without written consent from the study director.

Duplicates were taken of all samples and were stored at Harlan Laboratories Ltd., Füllinsdorf / Switzerland. The samples were not discarded without written consent from the study director.

RESULTS

The application formulations investigated during the study were found to comprise Acid Brown 425 in the range of 96.4% to 113.1% and, thus, the required content limit of ±20% with reference to the nominal content was met. The homogeneous distribution of Acid Brown 425 in the preparations was approved because single results found did not deviate more than 1.1% (acceptance criterion: <15%) from the corresponding mean.

In addition, the test item was found to be stable in application formulations when kept four hours at room temperature or eight days refrigerated due to recoveries which met the variation limit of ±10% from the time-zero (homogeneity) mean.

In conclusion, the results indicate the accurate use of the test item Acid Brown 425 and highly purified water as vehicle during this study. Application formulations were found to be homogeneously prepared and sufficient formulation stability under storage conditions was approved.
Duration of treatment / exposure:
Minimum 4 weeks (males) or approximately 7 weeks (females)
Frequency of treatment:
Once daily
Remarks:
Doses / Concentrations:
0, 100, 300 and 1000 mg/kg bw /day
Basis:
actual ingested
No. of animals per sex per dose:
11
Control animals:
yes, concurrent vehicle
Positive control:
not required
Observations and examinations performed and frequency:
VIABILITY/MORTALITY
Twice daily

CLINICAL SIGNS
Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy). Additionally females were observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.

FOOD CONSUMPTION
Pre-Pairing period days 1 - 8 and 8 - 14, gestation period days 0 - 7, 7 - 14 and 14 - 21 post coitum and lactation period days 1 - 4 post partum.
No food consumption was recorded during the pairing period.

BODY WEIGHTS
Recorded daily from treatment start to day of necropsy.

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

TERMINATION AND NECROPSY
Dams were sacrificed on day 5 post partum. If birth did not occur on the expected date (day 21 post coitum), the dam was sacrificed on day 25 post coitum.
At the scheduled sacrifice, all animals were weighed and sacrificed by an injection of sodium pentobarbital. All P generation animals were exsanguinated.
All parent animals were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred to establish, if possible, the cause of death. For the parent animals, special attention was directed at the organs of the reproductive system.

Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution.
The number of implantation sites and corpora lutea was recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites.

TISSUE PRESERVATION
The ovaries from all parental females were preserved in neutral phosphate buffered 4% formaldehyde solution.

HISTOTECHNIQUE
All organ and tissue samples to be examined by the principal investigator were processed, embedded and cut at an approximate thickness of 2 - 4 micrometers and stained with hematoxylin and eosin.

HISTOPATHOLOGY
Slides of all organs and tissues collected at terminal sacrifice from the animals of the control and high-dose groups were examined by the principal investigator. The same applied to all occurring gross lesions and to all animals, which died spontaneously or had to be terminated in extremis.

Histological examination of ovaries was carried out on any females that did not give birth.
A histopathology peer review was performed and the results included in the histopathology phase report.

Statistics:
The following statistical methods were used to analyze food consumption, body weights and reproduction data:

- Means and standard deviations of various data were calculated.
- The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
- Fisher's exact-test was applied if the variables could be dichotomized without loss of information.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
All animals survived the scheduled study period. Black colored feces were recorded in females of all groups treated with the test item from the start of dosing until necropsy. Severity of mid and high dose groups were increased. This finding was considered to be related to the treatment with the test item. In one female (no. 51) in the control group chromodacryorrhea of the left eye was recorded in the first four day of the pre-pairing period. No other clinical signs were noted in females at any dose level.
Mortality:
mortality observed, treatment-related
Description (incidence):
All animals survived the scheduled study period. Black colored feces were recorded in females of all groups treated with the test item from the start of dosing until necropsy. Severity of mid and high dose groups were increased. This finding was considered to be related to the treatment with the test item. In one female (no. 51) in the control group chromodacryorrhea of the left eye was recorded in the first four day of the pre-pairing period. No other clinical signs were noted in females at any dose level.
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Description (incidence and severity):
Discoloration of ovaries was recorded for two females in the 100 mg/kg body weight/day dose group and for one female in the 300 mg/kg body weight/day dose group. This finding was considered to be incidental
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
All findings recorded were considered to be within the range of normal background alterations.
Histopathological findings: neoplastic:
not examined
Details on results:
There were no test item-related effects found in any females in any dose group.
Treatment with the test item at the high-dose level caused slight reductions in food consumption, body weights and body weight gains in males observed during the pre-pairing. The main difference being between low and mid dose groups with marginal further decrease between mid and high dose groups despite the triplication of the dose.
There is indication of adaptive reactions with drecrease of effect over time.
The effects are considered test material related but not adverse, which is also suported by the absence of any effects in females even after prolonged exposure.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: No adverse effects at all
Remarks on result:
not measured/tested
Remarks:
Effect level not specified (migrated information)
Critical effects observed:
not specified
Conclusions:
There were no test item-related effects found in any females in any dose group.
Treatment with the test item at the high-dose level caused slight reductions in food consumption, body weights and body weight gains in males observed during the pre-pairing. The main difference being between low and mid dose groups with marginal further decrease between mid and high dose groups despite the triplication of the dose. There is indication of adaptive reactions with drecrease of effect over time. The effects are considered test material related but not adverse, which is also suported by the absence of any effects in females even after prolonged exposure.

No effects on mating performance, fertility, corpora lutea count or duration of gestation were observed at any dose level.

No test item-related findings were noted in pups up to day 4 post partum at any dose level.

Based on the evaluation of effects on food consumption, body weight and/or body weight gain in males the NOAEL for systemic toxicity for males was considered to be 1000 mg/kg body weight/day and the NOEL is 1000 mg/kg body weight/day for females.
Based on the absence of effects on fertility rate, mating performance, number of pups born and post natal loss of pups up to day 4 post partum the NOEL for reproduction toxicity was considered to be 1000 mg/kg body weight/day.
Executive summary:

This study is a valid investigation of the toxicological effects resulting from repeated oral-gavage administration of the test item Acid Brown 425 to rats. The test material was administered in highly purified water as vehicle at dosages of 100, 300, and 1000 mg/kg body weight/day, and controls received the vehicle only. The test material was administered to male rats for 28 days (except for one male up to 34 days) and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum (between 40 and 53 days for non-pregnant females and between 41 and 54 for dams).

Black colored feces seen in all test item-treated animals were considered to be a result of the staining properties of the dark brown test item.

There were no test item-related effects found in any females in any dose group.

Treatment with the test item at the high-dose level caused slight reductions in food consumption, body weights and body weight gains in males observed during the pre-pairing. The main difference being between low and mid dose groups with only marginal further decrease between mid and high dose groups despite the trippling of the dose. There is indication of adaptive reactions with drecrease of effect over time. The effects are considered test material related but not adverse, which is also suported by the absence of any effects in females even after prolonged exposure.

No effects on mating performance, fertility, corpora lutea count or duration of gestation were observed at any dose level.

No test item-related findings were noted in pups up to day 4 post partum at any dose level.

Based on the evaluation of effects on food consumption, body weight and/or body weight gain in males the NOAEL for systemic toxicity for males was considered to be 1000 mg/kg body weight/day and the NOEL is 1000 mg/kg body weight/day for females.

Based on the absence of effects on fertility rate, mating performance, number of pups born and post natal loss of pups up to day 4 post partum the NOEL for reproduction toxicity was considered to be 1000 mg/kg body weight/day.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
2 (valid with restriction)
the available study is a screening assay with reduced investigational program

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
repeated dose toxicity: inhalation
Data waiving:
exposure considerations
Justification for data waiving:
other:
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
repeated dose toxicity: inhalation
Data waiving:
exposure considerations
Justification for data waiving:
other:
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1983
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: read across, no GLP, outdated design, limited number of parameters examined
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
GLP compliance:
no
Remarks:
pre.dates GLP regulation
Limit test:
yes
Species:
rat
Strain:
other: Crl:CD(SD) BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Ltd. Manston Road, Margate, UK
- Age at study initiation: 5 weeks
- Weight at study initiation: 148 -245 g
- Fasting period before study: none
- Housing: single stainless teel wire mesh
- Diet (e.g. ad libitum): ad lib
- Water (e.g. ad libitum): ad lib.
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 24
- Humidity (%): 55 - 88
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 / 12
Type of coverage:
occlusive
Vehicle:
water
Details on exposure:
TEST SITE
- Area of exposure: 24 cm² in the dorsolumbar region

- Type of wrap if used: occlusive
- Time intervals for shavings or clipplings: when needed

REMOVAL OF TEST SUBSTANCE
- Washing (if done): none, but the treated area was wiped aftr 6 hours of exposure
- Time after start of exposure: 6h

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 1000 mg/kg
- Concentration (if solution): 250mg/mL
- Constant volume and concentration used: yes: 4 mL/kg bw / day
- For solids, paste formed: yes/no


USE OF RESTRAINERS FOR PREVENTING INGESTION: no
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
4 weeks
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
1000 mg/kg
Basis:
nominal per unit body weight
Remarks:
Doses / Concentrations:
0 mg/kg
Basis:
nominal per unit body weight
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: No sign of toxicity was een in an oral acute study in the same laboratory at a dose of 2000 mg/kg
Positive control:
no required
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: included in clinical examination

BODY WEIGHT: Yes
- Time schedule for examinations: before start, then weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, weekly

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: week 3
- Anaesthetic used for blood collection: Yes (diethyl ether)
- Animals fasted: Yes (16 hrs)
- How many animals: all
- Parameters examined: haemoglobin (Hb), red blood cell count (RBC) and derived fndices:
packed cell volume (PCV), mean cell haemoglobin (MCH), mean cell haemoglobin concentration (MCHC), mean cell volume (MCV)
total and differential white blood cell count (WBC)
In addition, samples were withdrawn into 3.13% trisodium citrate anticoagulant for determination of prothrombin time (PT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: week 3
- Animals fasted: Yes (16 hrs)
- How many animals: all
- Parameters examined:
glutamate oxaloacetate transaminase (GOT)
alkaline phosphatase (Alk.P)
blood urea nitrogen (BUN)
bilirubin
glucose
calcium
sodium
potass;um
chloride
inorganic phosphate
creatinine
total protein
albumin
albumin/globulin ratio

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (full external and internal macroscopic examination)
Organ weights of: adrenal, kidneys, liver, testes
HISTOPATHOLOGY: Yes (treated & untreated skin, adrenals, kidneys, liver, gross leasions)
Other examinations:
none
Statistics:
yes, but no detail given
Clinical signs:
no effects observed
Description (incidence and severity):
One control male was found dead on day 22. All animals treated with Brown HH 469 survived the 4 week study period. There were no treatment-related clinical signs.
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
Slight temporary signs of irritation (redness, very slight edema) were seen in some treated animals. No irritation was detected in controls.
Mortality:
no mortality observed
Description (incidence):
One control male was found dead on day 22. All animals treated with Brown HH 469 survived the 4 week study period. There were no treatment-related clinical signs.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
One control male was found dead on day 22. All animals treated with the test material survived the 4 week study period. There were no treatment-related clinical signs
Slight redness of the treated skin site was noted in some females exposed to the test material on days 2-4 and in 1 male on days 23 -28.
Slightly increased redness (grade 2) was noted on the treated skin site on female 16 on days 22-29 and in female 20 on days 11-26.
Very slight oedema was also noted on female 20 on days 11-12. One female showed skin sores on its back probably due to the occlusive dressing.
No adverse skin redness was noted in control animals.

BODY WEIGHT AND WEIGHT GAIN
Overall body weight gains during the treatment period were considered to be similar in all animals.

FOOD CONSUMPTION
There were no adverse effects on food intake.

HAEMATOLOGY
There were no changes in the haematological parameters of treated animals that could be attributed to treatment with Brown HH 469.

CLINICAL CHEMISTRY
There were no changes in the clinical chemistry parameters that could be attributed to treatment with Brown HH 469.

ORGAN WEIGHTS

GROSS PATHOLOGY
The majority of treated and control skin sites were unremarkable at necropsy. A minor sore was noted in 1 female treated with the test material but this was an isolated finding. The main skin reactions were sores and fur loss (alopecia) on the abdomen due to the adhesive dressing, but this was not related to treatment. The only consistent treatment-related finding was a brown discolouration of the tail, probably due to contamination by the test article. There were no unusual findings in the internal organs examined at necropsy except in one control male which died from urinary tract obstruction.

HISTOPATHOLOGY: NON-NEOPLASTIC
Skin reactions in the treated skin site of control rats consisted primarily of low grade acanthosis. This was more noticeable in males than females. The skin sites exposed to the test material were generally similar to those of the controls.
One male and 1 female had erosions, but these were minor focal lesions, probably of traumatic origin, and the rest of the treated skin area was similar to controls. Overall, there was no evidence of any significant skin irritation due to repeated percutaneous application of the test article.
Histopathology findings in the selected tissues examined were generally infrequent and of a minor nature such as hyaline droplets in the kidney of males and leucocyte foci in the livers of both sexes. There were no findings of any unusual nature or incidence to suggest any systemic toxic effect due to test article administration.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed at all.
Critical effects observed:
not specified
Conclusions:
Dermal application of Brown HH 469 at adose level of 1000 mg/kg/day for 4 weeks produced no evidence of systemic toxicity.
Executive summary:

1.1 The study was performed to evaluate the toxicity of the test article, Brown HH 469, when administered by dermal application to intact skin of the rat for a period of 4 weeks.

1.2 Groups of Sprague Dawley rats, comprising 5 animals of each sex, were treated at dose levels of 0 and 1000 mg/kg/day.

1.3 One control animal was found dead on day 22. All animals treated with 1000 mg/kg/day Brown HH 469 survived the 4 week study period.

1.4 No treatment-related changes in clinical condition or behaviour occurred.

1.5 Similar overall body weight gains were recorded in both test and control groups.

1.6 Food consumption was similar in both groups.

1.7 There were no changes in the haematological and clinical chemistry parameters measured.

1.8 All organ weights and organ/body weight ratios were considered similar for treated and control animals.

1.9 The gross and histological appearance of the skin site treated with Brown HH 469 was similar to that of control animals.

1.10 There was no evidence of any systemic toxicity in organs examined at necropsy or in tissues selected for histological evaluation. 1.11 In conclusion, dermal application of Brown HH 469 at a dose level of 1000 mg/kg/day produced no evidence of systemic toxicity.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Klimisch 2: read across, pre-dates GLP, limited number of parameters examined

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1983
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: read across, no GLP, outdated design, limited number of parameters examined
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
GLP compliance:
no
Remarks:
pre.dates GLP regulation
Limit test:
yes
Species:
rat
Strain:
other: Crl:CD(SD) BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Ltd. Manston Road, Margate, UK
- Age at study initiation: 5 weeks
- Weight at study initiation: 148 -245 g
- Fasting period before study: none
- Housing: single stainless teel wire mesh
- Diet (e.g. ad libitum): ad lib
- Water (e.g. ad libitum): ad lib.
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 24
- Humidity (%): 55 - 88
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 / 12
Type of coverage:
occlusive
Vehicle:
water
Details on exposure:
TEST SITE
- Area of exposure: 24 cm² in the dorsolumbar region

- Type of wrap if used: occlusive
- Time intervals for shavings or clipplings: when needed

REMOVAL OF TEST SUBSTANCE
- Washing (if done): none, but the treated area was wiped aftr 6 hours of exposure
- Time after start of exposure: 6h

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 1000 mg/kg
- Concentration (if solution): 250mg/mL
- Constant volume and concentration used: yes: 4 mL/kg bw / day
- For solids, paste formed: yes/no


USE OF RESTRAINERS FOR PREVENTING INGESTION: no
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
4 weeks
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
1000 mg/kg
Basis:
nominal per unit body weight
Remarks:
Doses / Concentrations:
0 mg/kg
Basis:
nominal per unit body weight
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: No sign of toxicity was een in an oral acute study in the same laboratory at a dose of 2000 mg/kg
Positive control:
no required
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: included in clinical examination

BODY WEIGHT: Yes
- Time schedule for examinations: before start, then weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, weekly

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: week 3
- Anaesthetic used for blood collection: Yes (diethyl ether)
- Animals fasted: Yes (16 hrs)
- How many animals: all
- Parameters examined: haemoglobin (Hb), red blood cell count (RBC) and derived fndices:
packed cell volume (PCV), mean cell haemoglobin (MCH), mean cell haemoglobin concentration (MCHC), mean cell volume (MCV)
total and differential white blood cell count (WBC)
In addition, samples were withdrawn into 3.13% trisodium citrate anticoagulant for determination of prothrombin time (PT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: week 3
- Animals fasted: Yes (16 hrs)
- How many animals: all
- Parameters examined:
glutamate oxaloacetate transaminase (GOT)
alkaline phosphatase (Alk.P)
blood urea nitrogen (BUN)
bilirubin
glucose
calcium
sodium
potass;um
chloride
inorganic phosphate
creatinine
total protein
albumin
albumin/globulin ratio

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (full external and internal macroscopic examination)
Organ weights of: adrenal, kidneys, liver, testes
HISTOPATHOLOGY: Yes (treated & untreated skin, adrenals, kidneys, liver, gross leasions)
Other examinations:
none
Statistics:
yes, but no detail given
Clinical signs:
no effects observed
Description (incidence and severity):
One control male was found dead on day 22. All animals treated with Brown HH 469 survived the 4 week study period. There were no treatment-related clinical signs.
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
Slight temporary signs of irritation (redness, very slight edema) were seen in some treated animals. No irritation was detected in controls.
Mortality:
no mortality observed
Description (incidence):
One control male was found dead on day 22. All animals treated with Brown HH 469 survived the 4 week study period. There were no treatment-related clinical signs.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
One control male was found dead on day 22. All animals treated with the test material survived the 4 week study period. There were no treatment-related clinical signs
Slight redness of the treated skin site was noted in some females exposed to the test material on days 2-4 and in 1 male on days 23 -28.
Slightly increased redness (grade 2) was noted on the treated skin site on female 16 on days 22-29 and in female 20 on days 11-26.
Very slight oedema was also noted on female 20 on days 11-12. One female showed skin sores on its back probably due to the occlusive dressing.
No adverse skin redness was noted in control animals.

BODY WEIGHT AND WEIGHT GAIN
Overall body weight gains during the treatment period were considered to be similar in all animals.

FOOD CONSUMPTION
There were no adverse effects on food intake.

HAEMATOLOGY
There were no changes in the haematological parameters of treated animals that could be attributed to treatment with Brown HH 469.

CLINICAL CHEMISTRY
There were no changes in the clinical chemistry parameters that could be attributed to treatment with Brown HH 469.

ORGAN WEIGHTS

GROSS PATHOLOGY
The majority of treated and control skin sites were unremarkable at necropsy. A minor sore was noted in 1 female treated with the test material but this was an isolated finding. The main skin reactions were sores and fur loss (alopecia) on the abdomen due to the adhesive dressing, but this was not related to treatment. The only consistent treatment-related finding was a brown discolouration of the tail, probably due to contamination by the test article. There were no unusual findings in the internal organs examined at necropsy except in one control male which died from urinary tract obstruction.

HISTOPATHOLOGY: NON-NEOPLASTIC
Skin reactions in the treated skin site of control rats consisted primarily of low grade acanthosis. This was more noticeable in males than females. The skin sites exposed to the test material were generally similar to those of the controls.
One male and 1 female had erosions, but these were minor focal lesions, probably of traumatic origin, and the rest of the treated skin area was similar to controls. Overall, there was no evidence of any significant skin irritation due to repeated percutaneous application of the test article.
Histopathology findings in the selected tissues examined were generally infrequent and of a minor nature such as hyaline droplets in the kidney of males and leucocyte foci in the livers of both sexes. There were no findings of any unusual nature or incidence to suggest any systemic toxic effect due to test article administration.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed at all.
Critical effects observed:
not specified
Conclusions:
Dermal application of Brown HH 469 at adose level of 1000 mg/kg/day for 4 weeks produced no evidence of systemic toxicity.
Executive summary:

1.1 The study was performed to evaluate the toxicity of the test article, Brown HH 469, when administered by dermal application to intact skin of the rat for a period of 4 weeks.

1.2 Groups of Sprague Dawley rats, comprising 5 animals of each sex, were treated at dose levels of 0 and 1000 mg/kg/day.

1.3 One control animal was found dead on day 22. All animals treated with 1000 mg/kg/day Brown HH 469 survived the 4 week study period.

1.4 No treatment-related changes in clinical condition or behaviour occurred.

1.5 Similar overall body weight gains were recorded in both test and control groups.

1.6 Food consumption was similar in both groups.

1.7 There were no changes in the haematological and clinical chemistry parameters measured.

1.8 All organ weights and organ/body weight ratios were considered similar for treated and control animals.

1.9 The gross and histological appearance of the skin site treated with Brown HH 469 was similar to that of control animals.

1.10 There was no evidence of any systemic toxicity in organs examined at necropsy or in tissues selected for histological evaluation. 1.11 In conclusion, dermal application of Brown HH 469 at a dose level of 1000 mg/kg/day produced no evidence of systemic toxicity.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
42 mg/cm²
Study duration:
subacute
Species:
rat
Quality of whole database:
Klimisch 2: read across, pre-dates GLP, limited number of parameters examined

Additional information

Based on the results of a dermal toxicity study with an analogous substance the NOAEL value for subacute dermal toxicity was calculated to be 1000 mg/kg bw in the rat. This finding was confirmed in an oral screening study according to OECD guideline 421 in rats with the target substance.

In accordance with REACH “Column 1” in Annex VIII the route of exposure most relevant to humans needs to be chosen; in this case this is the dermal route.

The results lead to the conclusion that the analogous substance does not exert systemic toxic effects after subacute dermal exposure and thus does not have to be classified, because:

1)     The NOAEL for subacute dermal toxicity is 1000 mg/kg bw, and

2)     Exposure of humans via oral uptake or inhalation is considered unlikely taking into account the vapor pressure and the physical form (powder treated with anti-dusting agents, solution) of the substance when manufactured or used.

Therefore, it is concluded that extented testing of subacute inhalation and oral toxicity is not scientifically justified.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
recent guideline study under GLP

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
sole valid study

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
sole valid study

Justification for classification or non-classification

Due to the findings described in the subacute dermal toxicity study on an analogous substance (No target organ could be detected.) the test item does not have to be classified for specific organ toxicity (STOT). Based on the substances use pattern dermal exposure is the most relevant route of exposure. Exposure via inhalation or orally is not expected to occur in the industrial setting. Therefore, Acid Brown 425 does not have to be classified as toxic to specific target organs