Registration Dossier

Administrative data

Description of key information

rat 28 day dermal repeat dose study; NOAEL >=1000mg/kg bw/day

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

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

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 July 2015 to 22 October 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Performed in accordance to OECD and GLP compliant
Qualifier:
according to
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Deviations:
yes
Remarks:
The study integrity was not adversely affected by the deviations.
Qualifier:
according to
Guideline:
other: EC No 440/2008, B.9: "Repeated Dose (28 days) Toxicity (dermal)", 2008.
Deviations:
yes
Remarks:
The study integrity was not adversely affected by the deviations.
GLP compliance:
yes (incl. certificate)
Limit test:
yes
Species:
rat
Strain:
other: Rat: Crl:WI(Han) (outbred, SPF-Quality)
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:Charles River Deutschland, Sulzfeld, Germany.
- Weight at study initiation: Males: 198 - 235 grams; females: 172 - 215 grams
- Housing: During the acclimatization, treatment and recovery phase, animals were housed individually in Macrolon plastic cages (MIII type; height 18 cm.).
Sterilized sawdust was provided as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and paper as cage-enrichment (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom). During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cageenrichment or bedding material.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: At least 5 days before the start of treatment under laboratory conditions.
- Fasting- Animals were deprived of food overnight (with a maximum of 24 hours) prior to scheduled necropsy.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40 to 70%
- Air changes (per hr): 10 air changes/hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle

IN-LIFE DATES: From: To:
Type of coverage:
occlusive
Vehicle:
water
Remarks:
water (Elix)
Details on exposure:
TEST SITE
- Area of exposure: Dermal application.
- % coverage: The test item was applied in an area of approx. 10% of the total body surface, i.e. approx. 40 cm² for males and 30 cm² for females.
- Type of wrap if used: The test item formulation was held in contact with the skin with a dressing, consisting of a surgical gauze patch (Surgy 1D; 8 ply)
, successively covered with aluminum foil and Coban flexible bandage. A piece of Micropore tape was additionally used for fixation of the bandages in females only.
- Time intervals for shavings or clipplings: One day before treatment (day -1), an area of approximately 5x7 cm on the back of the animals was clipped. Whenever necessary (during the course of the study) the skin-area was re-clipped at least 3 hours before a next application. Care was taken to avoid abrading the skin.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): 6 hours, after which the dressing was removed and the skin cleaned of residual test item using a dry paper towel and/or water.

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): The test item was dosed at 5 mL/kg bw. Actual dose volumes were calculated weekly according to the latest body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Quantitative analysis was based on the analytical method validated for the test substance in project
508721.

Analytical conditions:
Column
Stationary phase: Acclaim surfactant analytical column
Dimensions: 150 mm x 4.6 mm i.d., dp = 5 µm
Brand: Dionex, Sunnyvale, CA, USA
Column temperature: 40°C + or - 1°C
Injection volume: 50 µl
Mobile phase: 75/25 (v/v) A/B
Mobile phase: A – acetonitrile
B – Ammonium acetate buffer pH 5.4, 0.2M
Flow: 1.0 ml/min
UVVIS detection: 588 nm


Accuracy of preparation
The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%).

A small response at the retention time of the test substance was observed in the chromatograms of the Group 1 formulation prepared for use. This response was considered not to derive from the test substance since a similar response was obtained in chromatograms of blank water. Maximum contribution to the other groups was 0.06% based on peak area.

Homogeneity
The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Stability
Group 2 and Group 4 formulations were stable when stored at room temperature under normal laboratory light conditions for at least 6 hours.
Duration of treatment / exposure:
Duration of treatment: 28 days/ Exposure period: 6 hours
Frequency of treatment:
once daily
Remarks:
Doses / Concentrations:
0 mg/kg
Basis:
nominal per unit body weight
Remarks:
Doses / Concentrations:
100 mg/kg
Basis:
nominal per unit body weight
Remarks:
Doses / Concentrations:
300 mg/kg
Basis:
nominal per unit body weight
Remarks:
Doses / Concentrations:
1000 mg/kg
Basis:
nominal per unit area
No. of animals per sex per dose:
5/animals/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
- Rationale for animal assignment (if not random): By computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean.
Positive control:
No data
Observations and examinations performed and frequency:
Observations
Clinical signs: At least once daily.
Mortality: Twice daily.
Body weights: On Day 1 prior to dosing and on Days 5 and 10.
Food consumption: Over days 1-5 and 5-10

Mortality / Viability
At least twice daily. Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7). The time of death was recorded as precisely as possible.

Clinical signs
At least twice daily predose and immediately after the exposure period and during the recovery period once daily, detailed clinical observations
were conducted for all animals. The time of onset, grade and duration of any observed signs were recorded. Signs were graded for severity and the maximum grade will be predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) were scored.

Functional Observations
During Week 4 of treatment, the following tests were performed in all recovery animals and main control animal nos. 34 and 35 and all Main Group 2 and 3 animals after bandage removal and observation for clinical signs:
- hearing ability, pupillary reflex and static righting reflex (score 0 =normal/present, score 1 = abnormal/absent).
- fore- and hind-limb grip strength will be recorded as the mean of three measurements (Series M4-10, Mark-10 Corporation, J.J. Bos, Gouda, The Netherlands.
- motor activity test (recording period: 1 hour under normal laboratory light conditions for individual animals, using a computerized monitoring system (Kinder Scientific LLC, Poway, USA)). Total movements and ambulations were reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or finer movement like grooming, weaving or movements of the head.

No potential treatment-related findings were noted in hearing ability, pupillary reflex, static righting reflex, grip strength and/or motor activity at the end of treatment, therefore no functional observations were performed during recovery period.
Sacrifice and pathology:
Necropsy
Animals surviving to the end of the observation period and all moribound animals were deeply anaesthetized using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) and subsequently exsanguinated and subjected to a full post mortem examination. Animals were deprived of food overnight (with a maximum of 24 hours) prior to scheduled necropsy. All animals assigned to the study were necropsied and descriptions of all macroscopic abnormalities recorded. Rats found dead were subjected to a full post mortem examination as soon as possible after death and always within 24 hours. Samples of the following tissues and organs were collected from all animals at necropsy and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands) The treated skin was fixated separately from the other tissues and organs:
Identification marks: not processed
Adrenal glands (Aorta)
(Brain [cerebellum, mid-brain, cortex])
(Caecum)
(Cervix)
(Clitoral gland)
(Colon)
(Duodenum)
(Epididymides)*
(Eyes with optic nerve [if detectable] and
Harderian gland)*
(Female mammary gland area)
(Femur including joint)
Heart
(Ileum)
(Jejunum)
Kidneys
(Larynx)
(Lacrimal gland, exorbital)
Liver
(Lung, infused with formalin)
(Lymph nodes - mandibular, mesenteric)
(Nasopharynx)
(Oesophagus)
(Ovaries)
(Pancreas)
(Peyer's patches [jejunum, ileum] if detectable)
(Pituitary gland)
(Preputial gland)
(Prostate gland)
(Rectum)
(Salivary glands - mandibular, sublingual)
(Sciatic nerve)
(Seminal vesicles)
(Skeletal muscle)
Skin: normal and treated
(Spinal cord -cervical, midthoracic, lumbar)
Spleen
(Sternum with bone marrow)
(Stomach)
Testes*
(Thymus)
(Thyroid including parathyroid [if detectable])
(Tongue)
(Trachea)
(Urinary bladder)
(Uterus)
(Vagina)
All gross lesions

* fixed in modified Davidson's solution, prepared at WIL Research Europe using Formaldehyde 37-40%, Ethanol, Acetic acid -glacial (all Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA). Tissues were transferred to formalin after fixation for at least 24 hours.

Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.





Statistics:
The following statistical methods were used to analyze the data:

- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for
the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied when the data could not be assumed to follow a normal distribution.
- The exact Fisher-test was applied to frequency data.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
Clinical signs:
no effects observed
Dermal irritation:
not specified
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
A blue discolouration of urine was observed but was most likely caused by the black color of the test item.
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
discolouration of skin, tail and kidneys considered to be caused by the dye. There was no microscopic correlate to these macroscopic findings.
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
Results
Accuracy, homogeneity and stability over 6 hours of formulations of test item in Water (Elix) were demonstrated by analyses.

There were no toxicologically relevant changes at determination of clinical appearance, performance of functional observations, body weight and food consumption measurements, or alterations during clinical laboratory investigations, macroscopic examination, organ weight determination and microscopic examination.

Observations
Unscheduled removal of (part of) the bandage from the treated skin area by the animals was observed among all dose groups, including the control group (details specified in the raw data). This may relate to both the study procedure and a response of the animals to irritating substances in these types of studies. The incidence of bandage removal in this study was not related to the administered dose. Therefore, this occurrence was considered to be related the study procedures. Since the total time of removal of bandage per group was generally less than 5% of the total exposure time period, it was considered that the incidences of bandage removal did not adversely affect the outcome or the integrity of the study.

The total time of removal of bandage per group in percentage of the total nominal exposure duration is as follows:
group males females
1 2% ± 1.4 % 1% ± 1.2 %
2 1% ± 1.0 % 2% ± 2.1 %
3 1% ± 1.9 % 1% ± 0.6 %
4 1% ± 0.6% 1% ± 1.5 %

Mortality
Four females were found dead during the study: i.e. two control females nos.36 and 39 (Recovery group) and two high dose females no. 54 (Main group) and no.60 (Recovery group) on Days 12, 24, 3 and 3 respectively. No definite cause of death could be determined from the sections examined.

As these unscheduled deaths occurred at the same incidence in the control and the high dose group females, these deaths were not considered to be test item-related, but probably resulted from the test procedure with occlusive bandages.

The animals which were found dead on Day 3 were replaced on Day 4 to complete the high dose group and were treated up to 28-days.

Clinical Signs
There were no clinical signs of toxicity noted over the 28-day observation period.

In all dose groups treated with test substance a blue discolouration of treated skin, other body parts and/or urine were noted in a dose related manner, most likely caused by black color of the test item. In all groups scabs, wounds, scales and/or focal erythema of the treated skin and/or other body parts were noted during the treatment period. The incidence of these findings was not related to the dose. Since these symptoms occurred at similar incidence over the dose groups, the local effects are considered not toxicologically significant.

Incidental findings noted in this study may be secondary to the burden of dermal treatment rather than indicative of systemic toxicity. At the incidence observed, these were considered signs of no toxicological significance.

Functional Observations
Hearing ability, pupillary reflex and static righting reflex were normal in all animals. A slightly lower grip strength noted in males at 300 mg/kg occurred in absence of a dose-related response, similar changes in the opposite sex and supportive clinical signs. Therefore, the changes in grip strength were considered to be of no toxicological relevance.

Motor activity was similar between treated and control groups. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.

Body Weights
Body weights and body weight gain of treated animals remained in the same range as controls over the 4-week study period.

Food Consumption
Food consumption before or after allowance for body weight was similar between treated and control animals.

Clinical Laboratory Investigations
Haematology
No toxicologically relevant changes occurred in haematological parameters of treated rats.

Changes in prothrombin time and activated partial thromboplastin time as noted in males and females at 1000 mg/kg were considered to be slight in nature and occurred in opposite direction for both sexes and/or occurred only at the end of the recovery period. Therefore these changes were considered not to represent a change of biological significance.

Clinical Biochemistry
No toxicologically relevant changes occurred in clinical biochemical parameters of treated rats.

One single control male showed high aspartate aminotransferase (ASAT) and alanine aminotransferase (ALAT) activity levels. Since this was only seen in a control animal, this was considered to have occurred by chance with on relation to the test item.

Any other statistically significant changes in clinical biochemistry parameters were slight in nature and/or occurred in absence of a dose related distribution and therefor considered to be not toxicologically relevant.

Pathology
Macroscopic Examination
The following test item-related macroscopic findings were present:
- bluish discolouration of the treated skin in 5/5 males and 5/5 females at 100 mg/kg, in 5/5 males and 5/5 females at 300 mg/kg and in 10/10 males and 12/12 females treated at 1000 mg/kg;
- bluish discolouration of the tail in 1/5 males at 100 mg/kg, in 5/5 males and 2/5 females at 300 mg/kg and in 10/10 males and 10/12 females treated at 1000 mg/kg;
- greenish discolouration of the kidneys 1/5 males at 100 mg/kg, in 3/5 males and 5/5 females at 300 mg/kg and in 7/10 males and 8/12 females treated at 1000 mg/kg.
There was no microscopic correlate to these macroscopic findings and the discolouration was considered to be caused by the black colour of the applied test item and therefore not toxicologically relevant.

The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain in this type of study.

Organ Weights
There were no test item-related alterations in organ weights.

Microscopic Examination
There were no test item-related microscopic observations.
All of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.












Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Critical effects observed:
not specified

Mortality

Four females were found dead during the study: i.e. two control females nos.36 and 39 (Recovery group) and two high dose females no. 54 (Main group) and no.60 (Recovery group) on Days 12, 24, 3 and 3 respectively. No definite cause of death could be determined from the sections examined. As these unscheduled deaths occurred at the same incidence in the control and the high dose group females, these deaths were not considered to be test item-related, but probably resulted from the test procedure with occlusive bandages. The animals which were found dead on Day 3 were replaced on Day 4 to complete the high dose group and were treated up to 28-days.

Clinical Signs

There were no clinical signs of toxicity noted over the 28-day observation period.

In all dose groups treated with test substance a blue discolouration of treated skin, other body parts

and/or urine were noted in a dose related manner, most likely caused by black color of the test item.

In all groups scabs, wounds, scales and/or focal erythema of the treated skin and/or other body parts

were noted during the treatment period. The incidence of these findings was not related to the dose.

Since these symptoms occurred at similar incidence over the dose groups, the local effects are

considered not toxicologically significant.

Incidental findings noted in this study may be secondary to the burden of dermal treatment rather than

indicative of systemic toxicity. At the incidence observed, these were considered signs of no

toxicological significance.

Functional Observations

Hearing ability, pupillary reflex and static righting reflex were normal in all animals.

A slightly lower grip strength noted in males at 300 mg/kg occurred in absence of a dose-related

response, similar changes in the opposite sex and supportive clinical signs. Therefore, the changes in

grip strength were considered to be of no toxicological relevance.

Motor activity was similar between treated and control groups. All groups showed a similar motor

activity habituation profile with a decreasing trend in activity over the duration of the test period.

Body Weights

Body weights and body weight gain of treated animals remained in the same range as controls over

the 4-week study period.

Food Consumption

Food consumption before or after allowance for body weight was similar between treated and control

animals.

Clinical Laboratory Investigations

Haematology

No toxicologically relevant changes occurred in haematological parameters of treated rats.

Changes in prothrombin time and activated partial thromboplastin time as noted in males and females

at 1000 mg/kg were considered to be slight in nature and occurred in opposite direction for both sexes

and/or occurred only at the end of the recovery period. Therefore these changes were considered not

to represent a change of biological significance.

Clinical Biochemistry

No toxicologically relevant changes occurred in clinical biochemical parameters of treated rats.

One single control male showed high aspartate aminotransferase (ASAT) and alanine

aminotransferase (ALAT) activity levels. Since this was only seen in a control animal, this was

considered to have occurred by chance with on relation to the test item.

Any other statistically significant changes in clinical biochemistry parameters were slight in nature

and/or occurred in absence of a dose related distribution and therefor considered to be not

toxicologically relevant.

Pathology

Macroscopic Examination

The following test item-related macroscopic findings were present:

- bluish discolouration of the treated skin in 5/5 males and 5/5 females at 100 mg/kg, in 5/5 males

and 5/5 females at 300 mg/kg and in 10/10 males and 12/12 females treated at 1000 mg/kg;

- bluish discolouration of the tail in 1/5 males at 100 mg/kg, in 5/5 males and 2/5 females at 300

mg/kg and in 10/10 males and 10/12 females treated at 1000 mg/kg;

- greenish discolouration of the kidneys 1/5 males at 100 mg/kg, in 3/5 males and 5/5 females at

300 mg/kg and in 7/10 males and 8/12 females treated at 1000 mg/kg.

There was no microscopic correlate to these macroscopic findings and the discolouration was

considered to be caused by the black colour of the applied test item and therefore not toxicologically

relevant.

The remainder of the recorded macroscopic findings were within the range of background gross

observations encountered in rats of this age and strain in this type of study.

Organ Weights

There were no test item-related alterations in organ weights.

Microscopic Examination

There were no test item-related microscopic observations.

All of the recorded microscopic findings were within the range of background pathology encountered in

rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or

histologic character of those incidental tissue alterations.

Conclusions:
From the results presented in this report a No Observed Adverse Effect Level (NOAEL) for K1600
black dye of at least 1000 mg/kg was established.
Executive summary:

Repeated dose (28-days) dermal toxicity study with K1600 black dye by daily exposure in the rat, followed by a 14-day recovery period.

The study was based on the following guidelines:

- EC No 440/2008, B.9: "Repeated Dose (28 days) Toxicity (dermal)", 2008.

- OECD 410, "Repeated Dose Dermal Toxicity: 21/28-day Study", 1981.

Based on the results of a 10-day range finding study, the dose levels for the 28-day toxicity study were selected to be 0, 100, 300 and 1000 mg/kg.

The test item, K1600 black dye formulated in water, was topically applied for approximately 6 ho urs/day daily for 28 days onto clipped skin on the back of SPF-bred Wistar rats. One control group and three treated groups were tested, each consisting of 5 males and 5 females. And two recovery groups were included at control and high dose level, each consisting of 5 males and 5 females. Chemical analyses of formulations were conducted once during the study to assess accuracy, homogeneity and stability over 6 hours.

The following parameters were evaluated: clinical signs daily; functional observation tests in week 4; body weight and food consumption weekly; clinical pathology and macroscopy at termination; organ weights and histopathology on a selection of tissues. Results:

Accuracy, homogeneity and stability over 6 hours of formulations of test item in Water (Elix) were demonstrated by analyses.

There were no toxicologically relevant changes at determination of clinical appearance, performance of functional observations, body weight and food consumption measurements, or alterations during clinical laboratory investigations, macroscopic examination, organ weight determination and microscopic examination.

Conclusion:

From the results presented in this report a No Observed Adverse Effect Level (NOAEL) for K1600 black dye of at least 1000 mg/kg was established.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 July 2015 to 22 October 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Performed in accordance to OECD and GLP compliant
Qualifier:
according to
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Deviations:
yes
Remarks:
The study integrity was not adversely affected by the deviations.
Qualifier:
according to
Guideline:
other: EC No 440/2008, B.9: "Repeated Dose (28 days) Toxicity (dermal)", 2008.
Deviations:
yes
Remarks:
The study integrity was not adversely affected by the deviations.
GLP compliance:
yes (incl. certificate)
Limit test:
yes
Species:
rat
Strain:
other: Rat: Crl:WI(Han) (outbred, SPF-Quality)
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:Charles River Deutschland, Sulzfeld, Germany.
- Weight at study initiation: Males: 198 - 235 grams; females: 172 - 215 grams
- Housing: During the acclimatization, treatment and recovery phase, animals were housed individually in Macrolon plastic cages (MIII type; height 18 cm.).
Sterilized sawdust was provided as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and paper as cage-enrichment (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom). During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cageenrichment or bedding material.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: At least 5 days before the start of treatment under laboratory conditions.
- Fasting- Animals were deprived of food overnight (with a maximum of 24 hours) prior to scheduled necropsy.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40 to 70%
- Air changes (per hr): 10 air changes/hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle

IN-LIFE DATES: From: To:
Type of coverage:
occlusive
Vehicle:
water
Remarks:
water (Elix)
Details on exposure:
TEST SITE
- Area of exposure: Dermal application.
- % coverage: The test item was applied in an area of approx. 10% of the total body surface, i.e. approx. 40 cm² for males and 30 cm² for females.
- Type of wrap if used: The test item formulation was held in contact with the skin with a dressing, consisting of a surgical gauze patch (Surgy 1D; 8 ply)
, successively covered with aluminum foil and Coban flexible bandage. A piece of Micropore tape was additionally used for fixation of the bandages in females only.
- Time intervals for shavings or clipplings: One day before treatment (day -1), an area of approximately 5x7 cm on the back of the animals was clipped. Whenever necessary (during the course of the study) the skin-area was re-clipped at least 3 hours before a next application. Care was taken to avoid abrading the skin.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): 6 hours, after which the dressing was removed and the skin cleaned of residual test item using a dry paper towel and/or water.

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): The test item was dosed at 5 mL/kg bw. Actual dose volumes were calculated weekly according to the latest body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Quantitative analysis was based on the analytical method validated for the test substance in project
508721.

Analytical conditions:
Column
Stationary phase: Acclaim surfactant analytical column
Dimensions: 150 mm x 4.6 mm i.d., dp = 5 µm
Brand: Dionex, Sunnyvale, CA, USA
Column temperature: 40°C + or - 1°C
Injection volume: 50 µl
Mobile phase: 75/25 (v/v) A/B
Mobile phase: A – acetonitrile
B – Ammonium acetate buffer pH 5.4, 0.2M
Flow: 1.0 ml/min
UVVIS detection: 588 nm


Accuracy of preparation
The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%).

A small response at the retention time of the test substance was observed in the chromatograms of the Group 1 formulation prepared for use. This response was considered not to derive from the test substance since a similar response was obtained in chromatograms of blank water. Maximum contribution to the other groups was 0.06% based on peak area.

Homogeneity
The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Stability
Group 2 and Group 4 formulations were stable when stored at room temperature under normal laboratory light conditions for at least 6 hours.
Duration of treatment / exposure:
Duration of treatment: 28 days/ Exposure period: 6 hours
Frequency of treatment:
once daily
Remarks:
Doses / Concentrations:
0 mg/kg
Basis:
nominal per unit body weight
Remarks:
Doses / Concentrations:
100 mg/kg
Basis:
nominal per unit body weight
Remarks:
Doses / Concentrations:
300 mg/kg
Basis:
nominal per unit body weight
Remarks:
Doses / Concentrations:
1000 mg/kg
Basis:
nominal per unit area
No. of animals per sex per dose:
5/animals/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
- Rationale for animal assignment (if not random): By computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean.
Positive control:
No data
Observations and examinations performed and frequency:
Observations
Clinical signs: At least once daily.
Mortality: Twice daily.
Body weights: On Day 1 prior to dosing and on Days 5 and 10.
Food consumption: Over days 1-5 and 5-10

Mortality / Viability
At least twice daily. Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7). The time of death was recorded as precisely as possible.

Clinical signs
At least twice daily predose and immediately after the exposure period and during the recovery period once daily, detailed clinical observations
were conducted for all animals. The time of onset, grade and duration of any observed signs were recorded. Signs were graded for severity and the maximum grade will be predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) were scored.

Functional Observations
During Week 4 of treatment, the following tests were performed in all recovery animals and main control animal nos. 34 and 35 and all Main Group 2 and 3 animals after bandage removal and observation for clinical signs:
- hearing ability, pupillary reflex and static righting reflex (score 0 =normal/present, score 1 = abnormal/absent).
- fore- and hind-limb grip strength will be recorded as the mean of three measurements (Series M4-10, Mark-10 Corporation, J.J. Bos, Gouda, The Netherlands.
- motor activity test (recording period: 1 hour under normal laboratory light conditions for individual animals, using a computerized monitoring system (Kinder Scientific LLC, Poway, USA)). Total movements and ambulations were reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or finer movement like grooming, weaving or movements of the head.

No potential treatment-related findings were noted in hearing ability, pupillary reflex, static righting reflex, grip strength and/or motor activity at the end of treatment, therefore no functional observations were performed during recovery period.
Sacrifice and pathology:
Necropsy
Animals surviving to the end of the observation period and all moribound animals were deeply anaesthetized using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) and subsequently exsanguinated and subjected to a full post mortem examination. Animals were deprived of food overnight (with a maximum of 24 hours) prior to scheduled necropsy. All animals assigned to the study were necropsied and descriptions of all macroscopic abnormalities recorded. Rats found dead were subjected to a full post mortem examination as soon as possible after death and always within 24 hours. Samples of the following tissues and organs were collected from all animals at necropsy and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands) The treated skin was fixated separately from the other tissues and organs:
Identification marks: not processed
Adrenal glands (Aorta)
(Brain [cerebellum, mid-brain, cortex])
(Caecum)
(Cervix)
(Clitoral gland)
(Colon)
(Duodenum)
(Epididymides)*
(Eyes with optic nerve [if detectable] and
Harderian gland)*
(Female mammary gland area)
(Femur including joint)
Heart
(Ileum)
(Jejunum)
Kidneys
(Larynx)
(Lacrimal gland, exorbital)
Liver
(Lung, infused with formalin)
(Lymph nodes - mandibular, mesenteric)
(Nasopharynx)
(Oesophagus)
(Ovaries)
(Pancreas)
(Peyer's patches [jejunum, ileum] if detectable)
(Pituitary gland)
(Preputial gland)
(Prostate gland)
(Rectum)
(Salivary glands - mandibular, sublingual)
(Sciatic nerve)
(Seminal vesicles)
(Skeletal muscle)
Skin: normal and treated
(Spinal cord -cervical, midthoracic, lumbar)
Spleen
(Sternum with bone marrow)
(Stomach)
Testes*
(Thymus)
(Thyroid including parathyroid [if detectable])
(Tongue)
(Trachea)
(Urinary bladder)
(Uterus)
(Vagina)
All gross lesions

* fixed in modified Davidson's solution, prepared at WIL Research Europe using Formaldehyde 37-40%, Ethanol, Acetic acid -glacial (all Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA). Tissues were transferred to formalin after fixation for at least 24 hours.

Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.





Statistics:
The following statistical methods were used to analyze the data:

- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for
the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied when the data could not be assumed to follow a normal distribution.
- The exact Fisher-test was applied to frequency data.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
Clinical signs:
no effects observed
Dermal irritation:
not specified
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
A blue discolouration of urine was observed but was most likely caused by the black color of the test item.
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
discolouration of skin, tail and kidneys considered to be caused by the dye. There was no microscopic correlate to these macroscopic findings.
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
Results
Accuracy, homogeneity and stability over 6 hours of formulations of test item in Water (Elix) were demonstrated by analyses.

There were no toxicologically relevant changes at determination of clinical appearance, performance of functional observations, body weight and food consumption measurements, or alterations during clinical laboratory investigations, macroscopic examination, organ weight determination and microscopic examination.

Observations
Unscheduled removal of (part of) the bandage from the treated skin area by the animals was observed among all dose groups, including the control group (details specified in the raw data). This may relate to both the study procedure and a response of the animals to irritating substances in these types of studies. The incidence of bandage removal in this study was not related to the administered dose. Therefore, this occurrence was considered to be related the study procedures. Since the total time of removal of bandage per group was generally less than 5% of the total exposure time period, it was considered that the incidences of bandage removal did not adversely affect the outcome or the integrity of the study.

The total time of removal of bandage per group in percentage of the total nominal exposure duration is as follows:
group males females
1 2% ± 1.4 % 1% ± 1.2 %
2 1% ± 1.0 % 2% ± 2.1 %
3 1% ± 1.9 % 1% ± 0.6 %
4 1% ± 0.6% 1% ± 1.5 %

Mortality
Four females were found dead during the study: i.e. two control females nos.36 and 39 (Recovery group) and two high dose females no. 54 (Main group) and no.60 (Recovery group) on Days 12, 24, 3 and 3 respectively. No definite cause of death could be determined from the sections examined.

As these unscheduled deaths occurred at the same incidence in the control and the high dose group females, these deaths were not considered to be test item-related, but probably resulted from the test procedure with occlusive bandages.

The animals which were found dead on Day 3 were replaced on Day 4 to complete the high dose group and were treated up to 28-days.

Clinical Signs
There were no clinical signs of toxicity noted over the 28-day observation period.

In all dose groups treated with test substance a blue discolouration of treated skin, other body parts and/or urine were noted in a dose related manner, most likely caused by black color of the test item. In all groups scabs, wounds, scales and/or focal erythema of the treated skin and/or other body parts were noted during the treatment period. The incidence of these findings was not related to the dose. Since these symptoms occurred at similar incidence over the dose groups, the local effects are considered not toxicologically significant.

Incidental findings noted in this study may be secondary to the burden of dermal treatment rather than indicative of systemic toxicity. At the incidence observed, these were considered signs of no toxicological significance.

Functional Observations
Hearing ability, pupillary reflex and static righting reflex were normal in all animals. A slightly lower grip strength noted in males at 300 mg/kg occurred in absence of a dose-related response, similar changes in the opposite sex and supportive clinical signs. Therefore, the changes in grip strength were considered to be of no toxicological relevance.

Motor activity was similar between treated and control groups. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.

Body Weights
Body weights and body weight gain of treated animals remained in the same range as controls over the 4-week study period.

Food Consumption
Food consumption before or after allowance for body weight was similar between treated and control animals.

Clinical Laboratory Investigations
Haematology
No toxicologically relevant changes occurred in haematological parameters of treated rats.

Changes in prothrombin time and activated partial thromboplastin time as noted in males and females at 1000 mg/kg were considered to be slight in nature and occurred in opposite direction for both sexes and/or occurred only at the end of the recovery period. Therefore these changes were considered not to represent a change of biological significance.

Clinical Biochemistry
No toxicologically relevant changes occurred in clinical biochemical parameters of treated rats.

One single control male showed high aspartate aminotransferase (ASAT) and alanine aminotransferase (ALAT) activity levels. Since this was only seen in a control animal, this was considered to have occurred by chance with on relation to the test item.

Any other statistically significant changes in clinical biochemistry parameters were slight in nature and/or occurred in absence of a dose related distribution and therefor considered to be not toxicologically relevant.

Pathology
Macroscopic Examination
The following test item-related macroscopic findings were present:
- bluish discolouration of the treated skin in 5/5 males and 5/5 females at 100 mg/kg, in 5/5 males and 5/5 females at 300 mg/kg and in 10/10 males and 12/12 females treated at 1000 mg/kg;
- bluish discolouration of the tail in 1/5 males at 100 mg/kg, in 5/5 males and 2/5 females at 300 mg/kg and in 10/10 males and 10/12 females treated at 1000 mg/kg;
- greenish discolouration of the kidneys 1/5 males at 100 mg/kg, in 3/5 males and 5/5 females at 300 mg/kg and in 7/10 males and 8/12 females treated at 1000 mg/kg.
There was no microscopic correlate to these macroscopic findings and the discolouration was considered to be caused by the black colour of the applied test item and therefore not toxicologically relevant.

The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain in this type of study.

Organ Weights
There were no test item-related alterations in organ weights.

Microscopic Examination
There were no test item-related microscopic observations.
All of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.












Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Critical effects observed:
not specified

Mortality

Four females were found dead during the study: i.e. two control females nos.36 and 39 (Recovery group) and two high dose females no. 54 (Main group) and no.60 (Recovery group) on Days 12, 24, 3 and 3 respectively. No definite cause of death could be determined from the sections examined. As these unscheduled deaths occurred at the same incidence in the control and the high dose group females, these deaths were not considered to be test item-related, but probably resulted from the test procedure with occlusive bandages. The animals which were found dead on Day 3 were replaced on Day 4 to complete the high dose group and were treated up to 28-days.

Clinical Signs

There were no clinical signs of toxicity noted over the 28-day observation period.

In all dose groups treated with test substance a blue discolouration of treated skin, other body parts

and/or urine were noted in a dose related manner, most likely caused by black color of the test item.

In all groups scabs, wounds, scales and/or focal erythema of the treated skin and/or other body parts

were noted during the treatment period. The incidence of these findings was not related to the dose.

Since these symptoms occurred at similar incidence over the dose groups, the local effects are

considered not toxicologically significant.

Incidental findings noted in this study may be secondary to the burden of dermal treatment rather than

indicative of systemic toxicity. At the incidence observed, these were considered signs of no

toxicological significance.

Functional Observations

Hearing ability, pupillary reflex and static righting reflex were normal in all animals.

A slightly lower grip strength noted in males at 300 mg/kg occurred in absence of a dose-related

response, similar changes in the opposite sex and supportive clinical signs. Therefore, the changes in

grip strength were considered to be of no toxicological relevance.

Motor activity was similar between treated and control groups. All groups showed a similar motor

activity habituation profile with a decreasing trend in activity over the duration of the test period.

Body Weights

Body weights and body weight gain of treated animals remained in the same range as controls over

the 4-week study period.

Food Consumption

Food consumption before or after allowance for body weight was similar between treated and control

animals.

Clinical Laboratory Investigations

Haematology

No toxicologically relevant changes occurred in haematological parameters of treated rats.

Changes in prothrombin time and activated partial thromboplastin time as noted in males and females

at 1000 mg/kg were considered to be slight in nature and occurred in opposite direction for both sexes

and/or occurred only at the end of the recovery period. Therefore these changes were considered not

to represent a change of biological significance.

Clinical Biochemistry

No toxicologically relevant changes occurred in clinical biochemical parameters of treated rats.

One single control male showed high aspartate aminotransferase (ASAT) and alanine

aminotransferase (ALAT) activity levels. Since this was only seen in a control animal, this was

considered to have occurred by chance with on relation to the test item.

Any other statistically significant changes in clinical biochemistry parameters were slight in nature

and/or occurred in absence of a dose related distribution and therefor considered to be not

toxicologically relevant.

Pathology

Macroscopic Examination

The following test item-related macroscopic findings were present:

- bluish discolouration of the treated skin in 5/5 males and 5/5 females at 100 mg/kg, in 5/5 males

and 5/5 females at 300 mg/kg and in 10/10 males and 12/12 females treated at 1000 mg/kg;

- bluish discolouration of the tail in 1/5 males at 100 mg/kg, in 5/5 males and 2/5 females at 300

mg/kg and in 10/10 males and 10/12 females treated at 1000 mg/kg;

- greenish discolouration of the kidneys 1/5 males at 100 mg/kg, in 3/5 males and 5/5 females at

300 mg/kg and in 7/10 males and 8/12 females treated at 1000 mg/kg.

There was no microscopic correlate to these macroscopic findings and the discolouration was

considered to be caused by the black colour of the applied test item and therefore not toxicologically

relevant.

The remainder of the recorded macroscopic findings were within the range of background gross

observations encountered in rats of this age and strain in this type of study.

Organ Weights

There were no test item-related alterations in organ weights.

Microscopic Examination

There were no test item-related microscopic observations.

All of the recorded microscopic findings were within the range of background pathology encountered in

rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or

histologic character of those incidental tissue alterations.

Conclusions:
From the results presented in this report a No Observed Adverse Effect Level (NOAEL) for K1600
black dye of at least 1000 mg/kg was established.
Executive summary:

Repeated dose (28-days) dermal toxicity study with K1600 black dye by daily exposure in the rat, followed by a 14-day recovery period.

The study was based on the following guidelines:

- EC No 440/2008, B.9: "Repeated Dose (28 days) Toxicity (dermal)", 2008.

- OECD 410, "Repeated Dose Dermal Toxicity: 21/28-day Study", 1981.

Based on the results of a 10-day range finding study, the dose levels for the 28-day toxicity study were selected to be 0, 100, 300 and 1000 mg/kg.

The test item, K1600 black dye formulated in water, was topically applied for approximately 6 ho urs/day daily for 28 days onto clipped skin on the back of SPF-bred Wistar rats. One control group and three treated groups were tested, each consisting of 5 males and 5 females. And two recovery groups were included at control and high dose level, each consisting of 5 males and 5 females. Chemical analyses of formulations were conducted once during the study to assess accuracy, homogeneity and stability over 6 hours.

The following parameters were evaluated: clinical signs daily; functional observation tests in week 4; body weight and food consumption weekly; clinical pathology and macroscopy at termination; organ weights and histopathology on a selection of tissues. Results:

Accuracy, homogeneity and stability over 6 hours of formulations of test item in Water (Elix) were demonstrated by analyses.

There were no toxicologically relevant changes at determination of clinical appearance, performance of functional observations, body weight and food consumption measurements, or alterations during clinical laboratory investigations, macroscopic examination, organ weight determination and microscopic examination.

Conclusion:

From the results presented in this report a No Observed Adverse Effect Level (NOAEL) for K1600 black dye of at least 1000 mg/kg was established.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/cm²
Study duration:
subacute
Species:
rat

Additional information

Repeated dose (28-days) dermal toxicity study with K1600 black dye by daily exposure in the rat, followed by a 14-day recovery period. The study was based on the following guidelines: - EC No 440/2008, B.9: "Repeated Dose (28 days) Toxicity (dermal)", 2008. - OECD 410, "Repeated Dose Dermal Toxicity: 21/28-day Study", 1981. Based on the results of a 10-day range finding study, the dose levels for the 28-day toxicity study were selected to be 0, 100, 300 and 1000 mg/kg. The test item, K1600 black dye formulated in water, was topically applied for approximately 6 ho urs/day daily for 28 days onto clipped skin on the back of SPF-bred Wistar rats. One control group and three treated groups were tested, each consisting of 5 males and 5 females. And two recovery groups were included at control and high dose level, each consisting of 5 males and 5 females. Chemical analyses of formulations were conducted once during the study to assess accuracy, homogeneity and stability over 6 hours. The following parameters were evaluated: clinical signs daily; functional observation tests in week 4; body weight and food consumption weekly; clinical pathology and macroscopy at termination; organ weights and histopathology on a selection of tissues.

Results

Accuracy, homogeneity and stability over 6 hours of formulations of test item in Water (Elix) were demonstrated by analyses. There were no toxicologically relevant changes at determination of clinical appearance, performance of functional observations, body weight and food consumption measurements, or alterations during clinical laboratory investigations, macroscopic examination, organ weight determination and microscopic examination.

From the results presented in this report a No Observed Adverse Effect Level (NOAEL) for K1600 black dye of at least 1000 mg/kg was established.

Justification for classification or non-classification

There were no toxicologically relevant changes at determination of clinical appearance, performance of functional observations, body weight and food consumption measurements, or alterations during clinical laboratory investigations, macroscopic examination, organ weight determination and microscopic examination. From the results presented in this report a No Observed Adverse Effect Level (NOAEL) for K1600 black dye of at least 1000 mg/kg was established. The classification criteria for STOT repeat exposure are not met.