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Administrative data

Description of key information

In a reliable combined repeated dose and reproductive/developmental toxicity screening test (OECD TG 422) with the  surrogate CAS number 75247-18-6 (read across approach, please refer to chapter 13 of the IUCLID, Assessment reports) by oral gavage in rats  no toxicity occurred up to the highest administered dose of 1000 mg/kg bw/day. The NOAEL was determined to be 1000 mg/kg bw/day. No target organ was identified. Since the acute dermal toxicity study revealed a LD50 of > 2500 mg/kg bw in the absence of local clinical signs and the skin irritating study indicated no irritating effects, the repeated dose dermal toxicity can be considered as low when compared with oral exposure. The inhalation route is not of relevance due to the physico-chemical properties of the test item. 

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
short-term repeated dose toxicity: oral
combined repeated dose and reproduction / developmental screening
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP and OECD testing guideline compliant study with well-characterized test material. Based on ECHA-guidance, a default reliability of 2 is assigned to account for read-across.
according to guideline
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. QA statement)
Limit test:
Details on test animals or test system and environmental conditions:
- Source: Harlan, Horst, The Netherlands
- Age at study initiation: (P) 11-12 weeks at the start of the treatment period
- Weight at study initiation: the initial body weight variation did not exceed ±20% of the mean weight for each sex
- Housing: Macrolon cages (4 rats/sex)
- Diet: Cereal-based (closed formula) rodent diet (Rat & Mouse No. 3 Breeding Diet, RM3) from a commercial supplier (SDS Special Diets Services, Witham, England), ad libitum
- Water: ad libitum
- Acclimation period: 5 days

- Temperature (°C): 22± 2°C with a maximum of 24.9 °C
- Humidity (%): 44.2% and not exceeding 65%
- Air changes (per hr): 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark

From 2012-05-01 to 2012-06-15

Route of administration:
oral: gavage
CMC (carboxymethyl cellulose)
1% in water
Details on oral exposure:
Dilutions of the test substance in the vehicle and the vehicle for dosing the controls were prepared weekly and stored in a refrigerator (2-10ºC), in portions sufficient for one day. During dosing the dose formulation was stirred continuously.

- Concentration in vehicle: The vehicle was 1% aqueous CMC, dosing volume was 15 mL/kg bw/day
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
Homogenicity and concentration of the test item in the dose formulations was assessed twice during the treatment period.
Duration of treatment / exposure:
46 days
Frequency of treatment:
once daily (on a single occasion, animals 15 and 21 of the control group and animals 37,41,43 and 47 in the low dose group were erroneously dosed twice.)
Doses / Concentrations:
100, 300 and 1000 mg/kg bw/day
actual ingested
No. of animals per sex per dose:
12 rats
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on the results of a 14-day range-finding study in rats
Observations and examinations performed and frequency:
- Time schedule: twice daily
- Cage side observations: mortality, clinical observations

- Time schedule: prior to the first exposure and at the end of the study as part of the functional observation battery (FOB)

- Time schedule for examinations: Body weights of male and female animals were recorded shortly before the start of
administration of the test item at randomization and at the start of the study (day 0). Males
were weighed weekly until sacrifice. Females were weighed once per week during the premating period. Mated females were
weighed on days 0, 7, 14 and 21 during presumed gestation and on day 0 and 4 of lactation.
Non-mated females were weighed once per week after the mating period. In addition, the
animals were weighed on their scheduled necropsy date in order to calculate the correct organ to body weight ratios.

Feed consumption was measured per cage over weekly intervals during the study, with exemption of the mating period, during which no feed consumption was registered.

Prior to sacrifice, 5 animals/sex/group were fasted overnight (water was freely available) and blood was taken from the aorta during necropsy, whilst under CO2/O2 anaesthesia. K3-EDTA was used as anticoagulant. In each sample the following determinations were carried out:
packed cell volume
red blood cell count
total white blood cell count
differential white blood cell counts (neutrophils, lymphocytes, eosinophils,
basophils, monocytes)
prothrombin time
thrombocyte count
mean corpuscular volume (MCV; calculated)
mean corpuscular haemoglobin (MCH; calculated)
mean corpuscular haemoglobin concentration (MCHC; calculated)
activated partial prothoplastin time (APTT)
red blood cell districution width (RDW)

Prior to sacrifice, 5 animals/sex/group were fasted overnight (water was freely available) and blood was taken from the aorta during necropsy, whilst CO2/O2 anaesthesia. Blood was collected in heparinized plastic tubes and plasma was prepared by centrifugation. The following measurements were made in the plasma:
alkaline phosphatase activity (ALP),
aspartate aminotransferase activity (ASAT),
alanine aminotransferase activity (ALAT),
gamma glutamyl transferase activity (GGT),
total protein,
ratio albumin to globulin (calculated),
glucose (fasting),
bilirubin (total),
cholesterol (total),
calcium (Ca),
sodium (Na),
potassium (K),
chloride (Cl),
inorganic phosphate (PO4),
bile acids.

During neuro-behavioural testing, the observer was unaware of the treatment of the animals. FOB and spontaneous motor activity were assessed in all study animals during the predose phase and in 5 animals/sex/group at the end of the study.
Sacrifice and pathology:
All surviving male and female parent animals were sacrificed by exsanguination from the abdominal aorta whilst under CO2/O2 anaesthesia at necropsy and then examined grossly for pathological changes. Male animals were sacrificed after the mating period. Female animals were sacrificed at or shortly after day 4 of lactation. A necropsy was performed on animals that died intercurrently (if not precluded by autolysis) or that had to be killed because they were moribund. Prior to preservation of organs/tissues, the following organ weights were recorded: adrenals, brain, heart, kidneys, liver, ovaries, prostate, seminal vesicles, spleen, thymus, thyroid, uterus. Samples of the following tissues and organs of all parent animals were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde; except for the testes which were preserved in Bouin's fixative:

- ovaries (after counting the corpora lutea)
- uterus (after counting of the implantation sites)
- testes
- epididymides
- seminal vesicles
- prostate
- all gross lesions
In addition of 5 animals/sex/group following organs were preserved:
bone marrow (femur)
brain (including sections of cerebrum, cerebellum, medulla/pons)
clitorial gland
coagulation gland
femur including joint
jejunum (including Peyer's patches)
mesenterial and axillary lymph nodes
peripheral nerve (sciatic or tibial)
pituitary gland
preputial gland
seminal vesicles (including coagulation gland)
skeletal muscle (thigh)
spinal cord (cervical, mid-thoracic and lumbar)
thyroid (including parathyroid)
urinary bladder
* Non glandular (“forestomach”) and glandular (fundus, pylorus) parts were examined

Microscopic examination was performed on the collected organs of all animals of the control (group 1) and high dose group (group 4). If treatment-related changes were observed in the high-dose group, the evaluation of these tissues/organs was extended to the intermediate-dose groups (2 and 3).
In addition, reproductive organs of males that failed to sire (did not mate or mated females were not pregnant) and females that were non-mated or non-pregnant, of the low- and mid-dose groups, were microscopically examined. Furthermore, organs showing gross lesions of animals of all groups were microscopically examined.
The resulting data were analyzed using the methods mentioned below. Other statistical tests were performed when considered appropriate. P < 0.05 was considered as a level of significance.
- Body weight, body weight gain, food consumption and organ weights data were subjected to one way analysis of variance (ANOVA), followed by Dunnett or Kruskal-Wallis analysis.
- Haematology and clinical chemistry parameters were subjected to one way ANOVA followed by Dunnett’s multiple comparison tests.
- Mortality data and data of the pathology of parent animals were evaluated by the Fisher’s exact probability test.
Details on results:
There was no test item related mortality. One male of the low dose group (male animal 44) was found dead and partly cannibalized on day 17 of dosing, during the mating period. The animal showed no clinical signs leading to its death. Histopathology revealed widespread blue particles (test item) in the lungs confirming faulty dosing of the animal. Male animal 34 in the low dose group revealed hunched posture during the study. At autopsy an abscess in the thorax was found, confirming faulty dosing of the animal. No treatment related clinical signs were observed in males during the premating, mating and postmating period. No treatment related clinical signs were observed in females during the premating, mating, gestation and lactation period.
In several animals the tip of tail was missing or showed encrustations. Upon healing of the wound, the tail was considered complete and no observations were recorded. Male animals 40 (low dose) and 80 (high dose) showed grunting on several occasions.

No effects on body weight or body weight change were observed in males and females throughout the study.

No effect on food consumption was observed in males and females throughout the study. In the post-mating period a decrease in mean food consumption (mg/kg bw /day) was observed in the mid and high dose group males. However, as this was not observed in the food consumption when expressed as mg/animal/day, this was not considered to be a treatment-related effect.

There were no effects on haematology parameters.

There were no effects on clinical chemistry parameters.

The results of the neurobehavioural observations and motor activity did not indicate any neurotoxic potential of CAS no. 75247-18-6 in rats.

In each dosing group 12 females were placed with 12 males, resulting in 9/12 mated females in the control group, 11/12 mated females in the low and mid dose groups and 12/12 mated females in the high dose group. One female in the control group (female 1) seemed not mated, but was pregnant and delivered a litter after all. For this female no insemination date has been established. Male and female fertility indices and the mating index were slightly lower in the control group. The mean number of mating days until insemination was comparable in all groups. The mean number of corpora lutea and the mean number of implantation sites was comparable in all groups. The mean pre-implantation loss showed large inter group variations, but no treatment related effects were observed. Mean prenatal loss was comparable in all groups. All females survived delivery, resulting in 9/12, 10/12, 11/12 and 12/12 litters in the control group, low dose, mid dose and high dose groups, respectively. All females delivered litters with liveborn pups. One litter in the low dose group comprised one stillborn pup amongst liveborn pups. There were no litters comprising stillborn pups only.

No effects on organ weights were observed in male and female animals.

At necropsy many animals treated with the test substance showed blue (or bluish/green) discoloration of the (contents of the) gastro-intestinal tract and in a few cases also at mesenteric lymph nodes. This was obviously due to the color of the test substance. One male of the low-dose group, no 34, had a green nodule (2 cm) in the thoracic cavity, which appeared to be an abscess. This was clearly caused by faulty dosing (which means that the gavage needle accidently had penetrated the esophagus wall), resulting in a local abscess with blue coloured particles present in the interstitial tissue. In one female of the low dose group, no .45, the vaginal aperture was missing, which accounted for the dilated uterus and vagina and the observation that this animal had not mated. The other gross changes were unremarkable.

As a standard procedure at necropsy the intestines were flushed with formalin to enable proper preservation of the mucosa. Consequently the blue discoloured contents were flushed out for a large part and therefore not present in the slides. In several animals some intraluminal blue content was still visible in slides of the intestines. It was noted at the microscopic evaluation, but it was omitted from the summarizing histopathology table because of its limited relevance. There was no clear evidence that the blue substance was able to pass the intestinal wall, because it was only found in the lumen of the intestinal tract but not in other organs or tissues. There were a few exceptions though: there were two cases of faulty dosing in the low dose group (animals 34 (see above) and 44 (with widespread blue particles in the lungs; this animal was found dead on day 17 of the study) and in the high dose group (animal 84 which had some blue particles in the surrounding adipose tissue of the mesenteric lymph nodes which could not be explained). Further, microscopic examination of the sampled organs and tissues did not reveal treatment related histopathological changes. The histopathological changes observed were about equally distributed amongst the different treatment groups or occurred in one or a few animals only. They were common findings in rats of this strain and age or occurred as individual chance findings. Therefore, they were not considered to be related to treatment.
Dose descriptor:
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: There were no test item-related findings up to and including the highest dose level of 1000 mg/kg bw/day.
Critical effects observed:
not specified

A homogeneous distribution of the test item in the dose formulation was ensured by means of continuous stirring prior to dosing. The achieved concentrations were generally considered acceptable.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
1 000 mg/kg bw/day
Study duration:

Additional information

Repeated dose toxicity, oral route (read across)

In a repeated dose toxicity study (BASF SE, 2012, OECD 422) CAS number 75247 -18 -6, was administered to Wistar rats (4 groups of 12 male and 12 female animals each) via oral gavage at dose levels of 0, 100, 300 or 1000 mg/kg bw/day. Female rats were dosed during a premating period (2 weeks) and during mating (1 week), gestation and lactation until postnatal day 4 (alltogether: 46 days of treatment) for female animals. Male rats were dosed during premating period, during mating and up to the day of sacrifice. The study comprised. Mortality, clincal signs, body weight and food consumption were assessed at regular intervals. At the end of the study functional observation battery and motor activity parameters as well as haematology and clinical chemistry parameters were determined. At necropsy, selected organs were weighed and the animals were examined macroscopically and histopathologically. Relevant reproductive parameteres and indicies were determined. Daily clinical observations during the premating, gestation and lactation period did not reveal any treatment-related changes in the animals`appearance, general condition or behaviour. No treatment-related effects were observed in mean body weight, body weight changes and food consumption of exposed animals throughout the study. Neurological testing did not indicate any neurotoxic potential of the test item. At macroscopic examination many animals treated with the test substance showed blue (or bluish/green) discoloration of the (contents of the) gastro-intestinal tract, which was flushed out for a large part when the intestines were flushed with formalin to enable proper preservation of the mucosa. This discoloration was due to the color or the test substance and considered to represent a passive effect. Further, microscopic examination of the sampled organs and tissues did not reveal treatment related histopathological changes. No effects on organ weights were observed in male and female animals. In absence of effects on body weight, food consumption and histopathology the No Observed Adverse Effect Level (NOAEL) for males and females was established at the high dose level of 1000 mg/kg bw/day.



Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The surrogate CAS number 75247-18-6 is used for a read-across to the target substance CAS number 28654-73-1.

Justification for classification or non-classification

Based on the results of the repeated dose toxicity testing of the substance CAS number 75247 -18 -6, the test item CAS number 28654 -73 -1 does not need to be classified and labelled for danger of serious damage to health by prolonged exposure (R48) according to Directive 67/548/EEC (DSD) for specific target organ toxicity after repeated exposure (STOT RE) according to Regulation (EC) No 1272/2008 (CLP).