Decyldimethylamine

Endpoint summary

Description of key information

Reliable data from several guideline studies on repeated dose toxicity are available for C10-DMA, C12-14-DMA, C16-DMA and C18-DMA. These studies were performed either according to OECD TG 407 (C12-14 DMA), to OECD TG 421 (C12-14 DMA) or according to OECD TG 422 (C10-DMA, C12-14 DMA, C16-DMA and C18-DMA). Currently a OECD TG 422 study is on-going on C18-DMA. In addition, an OECD TG 408 study is planned either on C12-14 DMA or C16 DMA.

In addition, the results are supported by read-across data from several DMAOs. A two-year study with C10-16 DMAO is currently used to cover the endpoint subchronic/chronic toxicity. The toxicity patterns of all these studies are comparable to each other: The NOAEL and NOEL for systemic effects experimentally obtained is between 50 and 150 mg/kg in the available studies, no classification according to Regulation (EC) No. 1272/2008 is warranted. As key value the most conservative value for the category members is selected.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

repeated dose toxicity: oral, other

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2022

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Principles of method if other than guideline:

The test item was administered by oral gavage in graduated doses to three groups of male and female rats. The males were dosed for 50 days, up to and including the day before scheduled sacrifice (this includes two weeks prior to mating, during mating period and approximately, two weeks post mating period).

Females were dosed throughout the treatment period. This includes two weeks prior to mating (with the objective of covering at least two complete oestrous cycles), the variable time to conception, the duration of pregnancy and up to and including the day before scheduled sacrifice (i.e., up to LD13).
Animals in the recovery groups were kept only for observations of reversibility, persistence or delayed occurrence of systemic toxic effects for 14 days of recovery period and these animals were not mated and consequently were not used for assessment of reproduction/developmental toxicity. The recovery period of the study started from the first scheduled kill of dams.

GLP compliance:

yes

Specific details on test material used for the study:

Test item : Genamin 10R 302 D

CAS No. : 1120-24-7

Chemical name (IUPAC) : N,N-dimethyl-decan-1-amine

Physical Appearance : Clear, colourless liquid

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Rat is the standard laboratory rodent species used for toxicity assessment and also recommended by various regulatory authorities.

The Wistar rat was selected due to the large amount of background data available for this strain.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Rats were housed in an environment controlled room. The temperature maintained during the experiment was between 19.2 to 24.3°C and relative humidity between 60 and 68 %. The photoperiod was a 12 hours light and 12 hours dark cycle. Adequate fresh air supply of 12.6– 12.9 air changes/hour was maintained in the experimental room. The maximum and minimum temperature in the experimental room was recorded once daily. The relative humidity in the experimental room was calculated daily from dry and wet bulb temperature recordings

Route of administration:

oral: gavage

Details on route of administration:

Route of test item administration was through oral gavage. The oral route was chosen because it provides an exaggerated model of the normal exposure in humans.

Vehicle:

corn oil

Details on oral exposure:

Males: The dose formulation was administered orally by gavage to the rats of the specific groups once daily at approximately the same time each day (varying by ± 3 hours) for 50 days which includes 2 weeks prior to mating, during mating and post-mating, up to and including the day before scheduled sacrifice.
Females: The dose formulations were administered orally by gavage to the specific group of rats once daily at approximately the same time each day (varying by ± 3 hours) for total of 42-59 Days which includes 2 weeks prior to the mating, during mating, pregnancy and up to LD 13.
The dose formulations were administered to the high dose recovery group of rats once daily at approximately the same time each day (varying by ± 3 hours).
The dose volume administered to each rat was at an equivolume of 5 mL/kg body weight throughout the study. The dose volume was adjusted based on the most recent body weight of individual rat.
Similarly, vehicle was administered to rats in the vehicle control and vehicle control recovery groups at an equivolume of 5 mL/kg bwt.
The vehicle and the test item were not administered for vehicle control recovery and high dose recovery groups, respectively for 14 days from the first scheduled kill of dams.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

During the conduct of this study, the prepared dose formulations were analysed for test item concentration prior to dosing on Day 1 and during 2nd month (Day 47) of the treatment period. The results were considered acceptable, as the mean percent recovery was in the range, 70% to 120.0% at each dose level and %RSD at each dose level was less than or equal to 20%.

Duration of treatment / exposure:

Daily

Frequency of treatment:

Males: The dose formulation was administered orally by gavage to the rats of the specific groups once daily at approximately the same time each day (varying by ± 3 hours) for 50 days which includes 2 weeks prior to mating, during mating and post-mating, up to and including the day before scheduled sacrifice.
Females: The dose formulations were administered orally by gavage to the specific group of rats once daily at approximately the same time each day (varying by ± 3 hours) for total of 42-59 Days which includes 2 weeks prior to the mating, during mating, pregnancy and up to LD 13.
The dose formulations were administered to the high dose recovery group of rats once daily at approximately the same time each day (varying by ± 3 hours).
The dose volume administered to each rat was at an equivolume of 5 mL/kg body weight throughout the study. The dose volume was adjusted based on the most recent body weight of individual rat.
Similarly, vehicle was administered to rats in the vehicle control and vehicle control recovery groups at an equivolume of 5 mL/kg bwt.
The vehicle and the test item were not administered for vehicle control recovery and high dose recovery groups, respectively for 14 days from the first scheduled kill of dams.

Dose / conc.:

25 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

150 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Main groups: 10/sex/dose
Recovery groups: 5/sex/dose

Control animals:

yes

Details on study design:

Group No. Group Colour of
cage card Dose
(mg/kg/day) Concent-ration
(mg/mL) Dose volume
(mL/kg) No. of
Rats Sex Rat Numbers
From To
Main Groups
G1 Vehicle control White 0 0 5 10
10 M
F Rab4141
Rab4151 Rab4150
Rab4160
G2 Low dose Yellow 25 5 5 10
10 M
F Rab4161
Rab4171 Rab4170
Rab4180
G3 Mid dose Green 50 10 5 10
10 M
F Rab4181
Rab4191 Rab4190
Rab4200
G4 Mid-intermediate Pink 100 20 5 10
10 M
F Rab4201
Rab4211 Rab4210
Rab4220
G5 High Red 150 30 5 10
10 M
F Rab4221
Rab4231 Rab4230
Rab4240
Recovery Groups
G1R Vehicle control recovery White 0 0 5 5
5 M
F Rab4241
Rab4246 Rab4245
Rab4250
G5R High dose recovery Red 150 30 5 5
5 M
F Rab4251
Rab4256 Rab4255
Rab4260
M: Male; F: Female; Rab: Prefix code for rat numbers
Note: Females were selected with typical 4-5 days oestrous cycles.

Observations and examinations performed and frequency:

10.1 Clinical Signs, Morbidity and Mortality
All rats were observed for morbidity and mortality twice daily i.e., once in the morning and once in the afternoon. As there were no clinical signs of toxicity, the observation for morbidity and mortality was carried out once in the morning during weekend and holidays.
10.1 Clinical signs
All rats were observed for clinical signs once daily during the treatment and recovery period. Frequent observations were performed (twice) due to clinical sign of salivation. On the days of scheduled detailed clinical examination, clinical signs (after dosing) was included as a part of detailed clinical observations except on Day 1 wherein detailed clinical examination was done prior to the treatment and observations for general clinical signs was done after dosing the animals.
10.2 Detailed Clinical Examination
Detailed clinical examination was done prior to the treatment on Day 1 and at weekly intervals thereafter (±1 day) during the treatment period.
During detailed clinical examination, all rats were observed for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g., lacrimation, piloerection, pupil size, unusual respiratory pattern), changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g., excessive grooming, repetitive circling or bizarre behaviour like self-mutilation, walking backwards).
10.3 Functional Observation Battery Tests (FOB)
The following neurological examinations were performed on LD 13 for randomly selected 5 main group parental females, on Day 50 for randomly selected 5 main group parental males and at the end of the recovery period (Day 64) for all the recovery group animals.
10.3.1 Home Cage Observations
Each rat was observed in the home cage for posture and for presence or absence of abnormal vocalizations, tremors and convulsions.
10.3.2 Observations during Removal of Animal from Home Cage and Handling
The objective of this phase of neurological examination was to observe the subject’s response to handling and to conduct other procedures of the FOB that can best be performed when the rat is being held. Each rat was observed for the following examinations:
- ease of removal from home cage
- handling reactivity
- palpebral closure
- eye examination
- piloerection
- lacrimation
- salivation
- skin/fur examination
- perineum wetness
- respiration
- muscle tone and
- extensor thrust response
The observations were recorded using scores/ranks.
10.3.3 Open Field Observation
Rat was placed (one at a time) in an open arena, on a flat surface with a clean absorbent paper and observed for at least 2 minutes. Absorbent paper was replaced for each group. During this observation period, the rat was evaluated as it moved about freely/unperturbed and the following observations were made and was recorded using score/ranks:
- gait
- posture
- tremors
- mobility score
- arousal level
- clonic or tonic movements
- stereotypic behaviour
- bizarre behaviour
- urination
- defecation
- rearing
- abnormal vocalizations
10.3.4 Functional Tests
Functional testing included motor activity, sensory evaluation, landing hindlimbs footsplay and measurement of grip performance.
10.3.4.1 Motor Activity
The motor activity of rats was measured using an automated animal activity measuring system (Make: Columbus Instruments) equipped with a computer analyzer. Each rat was individually placed in the activity cages of the instrument. The rats were monitored for 30 minutes. During this motor activity measurement session, parameters viz., the stereotypic time (small movements) in seconds, the ambulatory time (large ambulatory movement) in seconds, distance travelled in inches and resting time in seconds were monitored. The Opto-Varimex 5 motor activity measurement system provided the data analysed at 10 minutes interval and the same was reported.
10.3.4.2 Sensory Reactivity Measurements
After the 2 minutes (approximately) observation period, while the rat was in the open field arena, the following tests were conducted. The rat was allowed to move freely in the open field box for these tests but positioned in the box by the observer in order to administer stimulus. During sensory reactivity measurements, rats were observed for following and the observations were recorded using scores/ranks.
- approach response
- touch response
- click response
- tail-pinch response
- pupil response
- aerial righting reflex

10.3.4.3 Landing Hindlimbs Footsplay
The landing hindlimbs footsplay was assessed by dropping the rat on to a horizontal surface of the table top from a short height and measured the distance between the hind feet upon landing. The heel portion of each hind foot of each rat was gently pressed to an ink pad just prior to testing. The rat was suspended in a prone position and then dropped from a height of approximately 30 cm on a SOP format, which contained the details such as study no., animal no, group, sex and date.
A clean recording SOP format was used for each rat. A total of 3 readings (Trial 1, Trial 2 and Trial 3) were recorded for each rat and average of 3 footsplay values are presented in the report along with the individual footsplay values.
10.3.4.4 Grip Performance
Hindlimbs and forelimbs grip performance was tested using dual grip strength meter (Model: Columbus Instruments). Three trials (Trial 1, Trial 2 and Trial 3) were conducted for each rat i.e., three trials each for forelimbs and hindlimbs. Average of three trials for both forelimbs and hindlimbs were calculated and presented in the report along with the individual grip strength values.
10.3.5 Physiological Observations
Body temperature (rectal temperature) was measured in degree Celsius (°C) using digital thermometer.
At the end of the functional test, body weight of each rat was measured.
10.4 Body Weights
i) Individual body weight of males were recorded on Day 1 and at weekly (±1 Day) intervals thereafter. Individual body weights of females were recorded on Day 1 and at weekly intervals thereafter till cohabitation (till mating confirmation) with males.

10.5 Food Consumption
Food consumption (g) was measured at weekly intervals (±1 Day) during treatment and recovery period.
Food consumption was not measured during the cohabitation period.

Sacrifice and pathology:

10.10.1 Necropsy
All adult animals and pups were subjected to detailed necropsy and findings were recorded. The adult animals killed at term were fasted overnight (water allowed), weighed and exsanguinated under isoflurane anesthesia. All the surviving pups were necropsied on LD 13 and findings were recorded. Particular attention was paid to the external genitals which were examined for signs of altered development. Dead pups were examined for possible defects and/or cause of death.
For apparently non-pregnant rats, the uteri were stained with 10% aqueous ammonium sulphide (Salewski staining method) to identify the peri-implantation loss of embryos (by staining the implantation sites) for confirmation of pregnancy.
The number of implantation sites were recorded for all the dams.
10.10.2 Tissue Collection
On completion of the gross pathology examination, the tissues/organs listed in the below table were collected and preserved in 10% neutral buffered formalin from all adults (parents) including recovery groups animals unless otherwise specified. The organ weights as percentage of body weights were determined and presented in the report.
Tissue/organ Organ
Weighing Collection and
Preservation Microscopic Examination
Gross Lesion/ Mass/ Nodule X X
Cervix X X
Epididymis X X X
Ovary X X X
Oviduct X X
Uterus1 X X X
Vagina X X
Gland, Prostate2 X X X
Gland, Seminal vesicle/ Gland, Coagulating2 X X X
Testis3 X X X
Gland, Thyroid/ Gland, Parathroid4 X X X
Muscle, Levator ani/ Bulbospongiosus X X
Gland, Bulbourethral X X
Penis, Glans X X
1: Weighed along with cervix
2: Prostate and seminal vesicles with coagulating glands were weighed as a whole; subsequently prostate was separated and weighed. The derived weight was presented for the seminal vesicles and coagulating glands
3: Collected in modified Davidson’s fluid
4: Weighed after fixation
X: Activity performed.
In addition, the following tissues were collected, weighed and preserved from 5 males and 5 females randomly selected from the main groups and all animals from the recovery groups. Organ weights as percentage of body weights were determined and are presented in the report.
Tissue/organ Organ
Weighing Collection and
Preservation Microscopic Examination
Brain (cerebrum, cerebellum, medulla oblongata and pons) X X X
Bone marrow, Femur5 X X
Bone, Femur/ Joint, Femorotibial9 X X
Bone marrow, Sternum X X
Bone, Sternum 9 X X
Large intestine, Cecum X X
Large intestine, Colon X X
Small intestine, Duodenum X X
Eye6 X X
Muscle, Skeletal X X
Gland, Adrenal X X X
Gland, Mammary X X
Gut-associated lymphoid tissue X X
Heart X X X
Small intestine, Ileum X X
Small intestine, Jejunum X X
Kidney X X X
Liver X X X
Lung7 X X
Lymph node, Mandibular X X
Lymph node, Mesenteric X X
Gland, Pituitary8 X X X
Large intestine, Rectum X X
Nerve, Sciatic X X
Spinal cord X X
Spleen X X X
Stomach X X
Thymus X X X
Trachea X X
Urinary bladder7 X X
X: Activity performed.
5: Prepared from femur marrow and stained using Giemsa stain.
6: Collected in Davidson’s fluid
7: Inflated with 10 % Neutral Buffered Formalin before immersion in fixative
8: Weighed after formalin fixation
9 : Decalcified prior to sectioning
On LD 13, thyroid gland was collected from available one randomly selected male and female pup from each litter and preserved in 10% NBF for the histopathological examination. The thyroid weighing for the pups was performed after fixation.
10.10.3 Histopathology
Tissues/organs collected from randomly selected 5 males and 5 females in the control and high dose groups (including reproductive organs) were examined microscopically for histopathological changes. Histopathological examination of the testes also involved qualitative assessment of the stages of spermatogenesis.
The reproductive organs (including thyroid/parathyroid) were not suspected of showing test item related changes in high dose group and hence the reproductive tissues were not examined in the remaining 5 animals of control and high dose groups and also in all respective lower dose group rats and recovery dose group rats.
Further, liver in males was suspected of showing test item related histopathological changes in the high dose group (G5) and were also examined in the five randomly selected lower dose group rats and all recovery group rats.
The reproductive organs of non-pregnant animals were also examined in all the dose groups.
All gross lesions were examined in all the groups.
The tissues were processed for routine paraffin embedding and 4 – 5 micron thickness sections were stained with Mayer’s Haematoxylin Eosin stain. In addition, testes were sectioned at 3-4 µm and stained with PAS reagent and haematoxylin to aid in qualitative assessment of spermatogenesis. Unused tissues were archived.

Statistics:

Data was captured using the ProvantisTM laboratory information management system (LIMS).
Parameters such as body weight, body weight change, body temperature, hindlimbs footsplay, grip performance, food consumption, organ weights, organ weight ratios (organ to body weight and organ to brain weight), laboratory Investigations – Haematology, Coagulation & Clinical Chemistry, oestrous cycle, pre-coital interval , ano-genital distance, post implantation loss (%), no. of implantations, mean litter size, sex ratio, survival index, gestation length (days), pups data and transferred (motor activity, thyroid profile) data was evaluated using the Levene Test for homogeneity of variances and the Shapiro-Wilks Test for normality of distributions. When data found to be homogeneous and of normal distribution, was analysed by analysis of variance (ANOVA), when data found to be nonhomogeneous or of nonnormal, the data was subjected for transformation and ANOVA was performed on transformed data. When ANOVA found to be significant, pairwise comparisons of treated groups to the control group was made using a parametric test, Dunnett, to identify statistical differences.
Data like mating and fertility indices were analysed using Chi-square test. When Chi-square is found to be significant, pairwise comparisons of treated groups to the control group was made using a Fisher Exact test, to identify statistical difference in ProvantisTM built-in statistical tests.
For two groups, the comparisons of mean between treatment and control group was done using student’s t-test.
Descriptive statistics Mean, SD, Percentages & Numbers was presented by Treatment group and Day.
All hypothesis testing was carried out at the 5% (2-sided) significance level unless otherwise specified. Significant differences are designated throughout the report as below:
*: Statistically significant difference from the control group at p < 0.05

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

The transient clinical sign of slight salivation was observed in all animals soon after test item administration from treatment Day 13 to end of treatment. Nevertheless, the symptom subsided within a few minutes and the rats were found to be normal. There were no clinical signs observed at 25, 50 and 100 mg/kg/day.
There was no mortality observed at any of the doses tested. There were no abnormalities observed in pups.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There was no effect on the mean fasting body weights and reproductive organ weights of adult rats at all the dose levels compared to their control counter parts in both the sexes. There were no significant changes in thyroid weight of pups on lactation Day 13. Administration of test item resulted in higher liver weight in parental males at all dose levels and was associated with hepatocyte hypertrophy, histologically at dose level of ≥ 100 mg/kg/day. However, the weight increase was not dose related. The liver weight remained unaffected in recovery rats. Females treated with the test item did not show any liver specific effects.

Gross pathological findings:

no effects observed

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Microscopically, hepatocyte hypertrophy (minimal) was noted in 100 mg/kg/day (1/5) and 150 mg/kg/day (1/5) males. The alteration was not accompanied by any test item attributed degenerative/inflammatory response. Hepatocyte hypertrophy was not observed in recovery rats. Female rats did not show any liver specific changes.
There were no test item-related histopathological changes noted in reproductive tissues of the parental male/female rats. The qualitative assessment of spermatogenesis in testes did not reveal any changes in the examined rats.
Reproductive tissues from non-pregnant rats did not show any microscopic changes.
There were no test item-related microscopic changes in thyroid gland of 13-Day old pups.

Key result

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effect found up to the highest dose level.

Critical effects observed:

no

Conclusions:

The results of the study indicated that the oral administration of test item GENAMIN 10 R 3202 D for 2 weeks prior to mating, during mating, and post mating did not cause any toxicological effect on general health, body weights and food consumption in males at 25, 50, 100 and 150 mg/kg/day and at all the tested doses in females. There were no mortalities observed during the treatment at all the tested doses.
At 150 mg/kg/day, the transient clinical sign of slight salivation was observed in all animals soon after test item administration from treatment Day 13 to end of treatment. There were no clinical signs observed at 25, 50 and 100 mg/kg/day.
The transient clinical sign of slight salivation observed was subsided within few minutes. This finding may be attributed to local oral mucosa irritation. It is well known that salivation is often observed in gavage studies and may be a reaction to the taste or irritation of the test article rather than an indication of toxicity. Therefore, it was considered that the transient salivation observed in this study was of no toxicological significance.
The test item administration did not reveal any treatment related changes in the hematology, coagulation, clinical chemistry, urinalysis parameters, terminal fasting body weights and gross pathology endpoints in the parental rats of either sex. There were no developmental and non-developmental gross findings noted in male or female pups.
Microscopically, hepatocyte hypertrophy was noted in ≥100 mg/kg/day males and was accompanied by higher liver weight at all the doses (non-dose related). The liver findings showed complete reversal at the end of 14-day recovery phase.
There were no test item-related histopathological changes noted in reproductive tissues in any of the parental male and female rats. The qualitative assessment of spermatogenesis in testes did not reveal any changes in the examined rats.
Reproductive tissues from non-pregnant rats did not show any microscopic changes.
There were no test item-related microscopic changes in thyroid gland of 13-Day old pups.
No Observed Adverse Effect Level

As there were no treatment related effects on systemic, reproduction and fertility parameters up to and including the highest dose tested 150 mg/kg bwt/day, the No Observed Adverse Effect Level (NOAEL) for Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test by Oral Gavage in Wistar rats for the test item Genamin 10 R 302 D is determined to be 150 mg/kg bwt/day under the test conditions and doses employed.

Executive summary:

The purpose of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in Wistar rats by oral gavage was to determine the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. Further, this study provides initial information on possible effects of the test item on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus and parturition. This study also provides information on reversibility, persistence or delayed occurrence of systemic toxic effects, following 14 days post treatment.

The test item was weighed and suspended in vehicle [Corn oil] and administered at the graduated dose levels of 25, 50, 100 and 150 mg/kg bwt/day for low dose (G2), mid dose (G3), Mid-intermediate dose (G4) and High dose(G5)/high dose recovery (G5R) group rats, respectively. The rats in the vehicle control (G1)/vehicle control recovery (G1R) groups received vehicle alone. The dose volume administered was 5 mL/kg bwt/day. Each main group in the experiment comprised of 10 male and 10 female rats and recovery group comprised of 5 male and 5 female rats.

The dose formulations were administered once daily to a specific group of rats for two weeks prior to mating, during mating and post-mating periods (for males), during pregnancy and up to Lactation Day (LD) 13 for females.

The identity of the test item was provided by the Sponsor by a Certificate of Analysis (CoA). The authenticity of the test item was not determined at the test facility. The stability of the test item in the vehicle was established separately under Eurofins Advinus Study No. G22702 at 1 and 100 mg/mL. Based on the results, the test item was found to be stable in the vehicle for up to 48 hours when stored at room temperature.

During the conduct of this study, the prepared dose formulations were analysed for test item concentration prior to dosing on Day 1 and during 2^nd month (Day 47) of the treatment period. The results were considered acceptable, as the mean percent recovery was in the range, 70% to 120.0% at each dose level and %RSD at each dose level was less than or equal to 20%.

All rats were observed for clinical signs once daily. Body weight was recorded prior to the start of treatment on Day 1 and at weekly intervals thereafter. The body weights were also recorded at termination. Food consumption was recorded at weekly intervals except during the cohabitation period.

Neurological examinations were conducted for randomly selected 5 main group females on LD 13 and randomly selected 5 main group males on treatment day 51 and towards the end of recovery period (Day 64) for the recovery group animals.

Laboratory investigations such as hematology, coagulation, clinical chemistry and urinalysis were performed on randomly selected 5 parental males and females from each main group at the end of two weeks pre-mating period, towards the end of recovery period for all animals after overnight fasting. Thyroxine 4 (T4) and Thyroid Stimulating Hormone (TSH) analysis were performed in all main group males at termination, all dams on LD 13 and from available pups on LD 4 and 13.

At sacrifice, the parental males (Day 51), parental females (LD 14) and the recovery animals (Day 67) were subjected to detailed necropsy after overnight fasting (water allowed) and the study plan specified tissues were collected. The pups were sacrificed on LD 13 after examining the external genitals for signs of altered development.

Histopathology examination was carried out on the preserved organs from randomly selected 5 males and 5 females in the control and high dose groups (including reproductive organs) and on all gross lesions. Histopathological examination of testes included a qualitative assessment of stages of spermatogenesis. Thyroids were examined from all adult males and females and in LD13 pups. The reproductive organs were examined from the non-pregnant females.

 

Under the experimental conditions employed, the following results were obtained:

Clinical signs and Mortality: There were no clinical signs observed at 25, 50 and 100 mg/kg/day. At 150 mg/kg/day, the transient clinical sign of slight salivation was observed in all animals soon after test item administration from treatment Day 13 to end of treatment. There was no mortality observed at any of the doses tested.

The transient clinical sign of slight salivation observed was subsided within few minutes. This finding may be attributed to local oral mucosa irritation. It is well known that salivation is often observed in gavage studies and may be a reaction to the taste or irritation of the test article rather than an indication of toxicity. Therefore, it was considered that the transient salivation observed in this study was of no toxicological significance.

There were no mortalities observed during the treatment at all the tested doses. There were no abnormalities observed in pups.

Functional Observation Battery: No treatment-related neurological abnormalities were observed at any of the doses tested.

 

Body weights: The mean body weights and body weight gains were unaffected by the treatment at all the tested doses in both sexes.

Food consumption: Treatment did not affect the food consumption at any of the tested doses in either sex.

Haematology, Coagulation, Clinical chemistry and Urine Parameters: No test item-related changes were observed in the haematology, coagulation, clinical chemistry and urine parameters at all the doses tested in both sexes.

Hormone analysis: The thyroid stimulating hormone (TSH) and thyroxine (T4) levels in adult rats and pups remained unaffected by test item administration.

Terminal fasting body weights, organ weights and its ratios: There was no effect on the mean fasting body weights and reproductive organ weights of adult rats at all the dose levels compared to their control counter parts in both the sexes. There were no significant changes in thyroid weight of pups on lactation Day 13. Administration of test item resulted in higher liver weight in parental males at all dose levels and was associated with hepatocyte hypertrophy, histologically at dose level of ≥ 100 mg/kg/day. However, the weight increase was not dose related.  The liver weight remained unaffected in recovery rats. Females treated with the test item did not show any liver specific effects.

 

Gross and histopathology:

There were no test item-related gross lesions observed in both the sexes. There were no developmental and non-developmental gross findings noted in male or female pups.

Microscopically, hepatocyte hypertrophy (minimal) was noted in 100 mg/kg/day (1/5) and 150 mg/kg/day (1/5) males. The alteration was not accompanied by any test item attributed degenerative/inflammatory response. Hepatocyte hypertrophy was not observed in recovery rats. Female rats did not show any liver specific changes. 

There were no test item-related histopathological changes noted in reproductive tissues of the parental male/female rats. The qualitative assessment of spermatogenesis in testes did not reveal any changes in the examined rats.

There were no test item-related microscopic changes in thyroid gland of 13-Day old pups.

 

In view of the results observed:

As there were no treatment related effects on systemic, reproduction and fertility parameters up to and including the highest dose tested 150 mg/kg bwt/day, the No Observed Adverse Effect Level (NOAEL) for Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test by Oral Gavage in Wistar rats for the test item Genamin 10 R 302 D is determined to be 150 mg/kg bwt/day under the test conditions and doses employed.

Reference 2

Endpoint:

repeated dose toxicity: oral, other

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

For details on endpoint-specific justification, please see read-across justification document (category approach) in the linked category of dimethylalkylamines.

Frequency of treatment:

Daily

Organ weight findings including organ / body weight ratios:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

69 mg/kg bw/day (actual dose received)

Based on:

other: Read-across from C16-DMA; value converted based on MW

Sex:

male

Basis for effect level:

body weight and weight gain

Remarks on result:

other: Original data for the source: 100 mg/kg bw/d

Key result

Critical effects observed:

no

Executive summary:

The study used as source investigated the repeated oral toxicity of C16-DMA. The study results of the source compound were considered applicable to the target compound. Justification and applicability of the read-across approach (category approach) is outlined in the read-across report in the linked category of dimethylalkylamines.

Reference 3

Endpoint:

repeated dose toxicity: oral, other

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

For details on endpoint-specific justification, please see read-across justification document (category approach) in the linked category of dimethylalkylamines.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 124 mg/kg bw/day (actual dose received)

Based on:

other: Read-across from C12-14 DMA; value converted based on MW

Sex:

male/female

Basis for effect level:

other: No adverse effect up to the highest dose

Remarks on result:

other: Original data for the source: >= 150 mg/kg bw/d

Key result

Critical effects observed:

no

Executive summary:

The study used as source investigated the repeated oral toxicity of C12-14 DMA. The study results of the source compound were considered applicable to the target compound. Justification and applicability of the read-across approach (category approach) is outlined in the read-across report in the linked category of dimethylalkylamines.

Reference 4

Endpoint:

chronic toxicity: oral

Remarks:

combined repeated dose and carcinogenicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Basic data given: comparable to guidelines/standards.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

GLP compliance:

no

Remarks:

original study performed in 1979, prior to the adoption of GLP compliance standards. However, it was reviewed and found acceptable by the laboratory´s Quality Assurance Department in accordance with US FDA´s GLP Regulation of June 20, 1979.

Limit test:

no

Species:

rat

Strain:

other: Charles River

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding laboratories Inc., Portage, Michigan, USA
- Age at study initiation: 4 weeks
- Housing: individually
- Diet: Zeigler NIH-07diet containing test substance, ad libitum
- Water: ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-24
- Humidity (%): 46-70
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): Zeigler NIH-07

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

not indicated

Duration of treatment / exposure:

104 weeks

Frequency of treatment:

daily with the food

Remarks:

Doses / Concentrations:
0, 0.01, 0.1 and 0.2 % of the test compound (100% (w/v) active basis)
Basis:
nominal in diet

No. of animals per sex per dose:

60

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Doses were selected based on the results from a 13 week study which showed the high dose causing about a 5 to 8 % reduction in body weight.
- Concentrations in the diet of 0, 0.01, 0.1 and 0.2 % test compound (100% (w/v) active basis) correspond to 0, 4.24, 42.3, or 87.4 mg/kg bw/day for males and 0, 5.23, 52.6, or 107 mg/kg bw/day for females according to OECD (2006).

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly for the first 14 weeks of study, biweekly for the next 12 weeks and monthly thereafter.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/week: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

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: yes

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: once during pretest and at 3,6,12,16,19,22 and 24 months
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: all rats to be sacrificed at 52 weeks of the study and all rats at study termination
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: all

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: all rats to be sacrificed at 52 weeks of the study and all rats at study termination
- Animals fasted: No data
- How many animals: all

URINALYSIS: Yes
- Time schedule for collection of urine: all rats to be sacrificed at 52 weeks of the study and all rats at study termination
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Statistics:

Body weights, feed consumption, efficiency of feed utilization, clinical chemistry parameters and absolute and relative organ weights were compared by analysis of variance (one way classification), Bartlett´s test for homogeneity of variances, and at least significant differences criterion. If Bartlett´s test were significant, then pairwise comparisons were made by the Mann-Whitney U test. All statistical analyses were conducted at a 5 %, two-sided risk level, and each treatment group was compared with the main control group by sex.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

details see below

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

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
There were no significant differences in survival at 104 weeks. There were no significant, compound-related differences in mean feed consumption, clinical chemistry or ophthalmology.

BODY WEIGHT AND WEIGHT GAIN
The high dose animals demonstrated >10% decreases in mean body weight. Animals in the 0.1% test substance group also showed reduced body weight, but the reduction was not as strong as in the high dose group.

HISTOLOGY
There were no compound-related effects on histopathic examination. There was no evidence of a carcinogenic response after chronic dietary administration of the test substance.

Dose descriptor:

NOAEL

Effect level:

42.3 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: based on decreased mean body weight in the highest dose group.

Dose descriptor:

NOAEL

Effect level:

52.6 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: based on decreased mean body weight in the highest dose group.

Critical effects observed:

not specified

Conclusions:

Under the conditions of the test, no substance related effects were observed in male and female rats after oral exposure for 104 weeks. The NOAEL is 42.3 (males) or 52.6 (females) mg/kg bw/day.

Executive summary:

Male and female Charles River rats were given a diet containing 0, 0.01, 0.1 or 0.2 % test substance ad libitum for 104 weeks. No substance related effect was observed in any concentration tested. The NOAEL is 42.3 (male) or 52.6 (female) mg/kg bw/day based on decreased mean body weight in the highest dose group.

Reference 5

Endpoint:

chronic toxicity: oral

Remarks:

combined repeated dose and carcinogenicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

For details on endpoint specific justification please see read-across report in section 13 or find a link in cross-reference “assessment report”.

Reason / purpose for cross-reference:

assessment report

Reason / purpose for cross-reference:

read-across source

Details on study design:

-

Clinical signs:

no effects observed

Mortality:

no mortality observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

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
There were no significant differences in survival at 104 weeks. There were no significant, compound-related differences in mean feed consumption, clinical chemistry or ophthalmology.

BODY WEIGHT AND WEIGHT GAIN
The high dose animals demonstrated >10% decreases in mean body weight. Animals in the 0.1% test substance group also showed reduced body weight, but the reduction was not as strong as in the high dose group.

HISTOLOGY
There were no compound-related effects on histopathic examination. There was no evidence of a carcinogenic response after chronic dietary administration of the test substance.

Dose descriptor:

NOAEL

Effect level:

42.3 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: based on decreased mean body weight in the highest dose group.

Dose descriptor:

NOAEL

Effect level:

52.6 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: based on decreased mean body weight in the highest dose group.

Critical effects observed:

not specified

Executive summary:

The study used as source investigated chronic toxicity after oral exposure. The study results of the source compound were considered applicable to the target compound. Justification and applicability of the read-across approach (structural analogue) is outlined in the read-across report in section 13 or find a link in cross reference “assessment report”.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

good

System:

other: Body weight

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

Additional information

Toxicity after repeated oral application has been investigated with the following substances of the DMA category: C10-DMA, C12-14-DMA, C16-DMA. The studies are reliable (reliability 1 or 2) and were performed according to OECD TG 422 or 421. For one substance, C12-14-DMA, a study on subchronic toxicity according to OECD Guideline 407 is available. All available studies are gavage studies in rats with sesame or corn oil as vehicle.

Additionally, there are several studies for repeated dose toxicity after oral or dermal application available for DMAOs  in a read across procedure.

In the 28-day study (OECD TG 407, reliability 1) with C12-14-DMA no general toxicity was observed at a dose of 50 mg/kg bw/d. In the next higher dose (150 mg/kg bw/d), only marginal effects like rubbing of the snouts in the bedding material were observed. Lethality occurred at 300 mg/kg bw/d. The NOEL in this study was 50 mg/kg bw/d.

This NOEL was confirmed for the same substance with a reliable screening study for reproduction/developmental toxicity according to OECD guideline 421. Male and female rats were exposed for at least 28 days (males) or longer (females: 14 days prior to mating and during the mating period, pregnancy and lactation period). No effects were observed at 50 mg/kg bw/d, however, at 150 mg kg bw/d, the next higher dose group, lethality was already reported. Even at higher doses (up to 450 mg/kg bw/d) only lethality, but no specific toxicity effects were observed.

The newer screening study from 2022 performed with the same substance according to OECD TG 422 resulted in a NOAEL of 150 mg/kg bw/d which was the highest dose tested. No treatment related adverse effects on systemic, reproduction and fertility parameters were observed. In this study type male animals are exposed for 50 days (this includes two weeks prior to mating, during mating period and approximately, two weeks post mating). Female rats are dosed throughout the treatment period. This includes two weeks prior to mating, the variable time to conception, the duration of pregnancy and up to and including the day before scheduled sacrifice (i.e., up to LD13). No mortality was observed at any dose level. In the 14-week DRF study for the OECD-422 study adverse effects at 300 mg/kg be/d were observed including reduced food intake, reduced organ weights, changes in clinical chemistry parameters and irritation in GI tract.

The results from the OECD-422 study for C10-DMA support the findings from the screening study for C12-14 DMA. The NOAEL is 150 mg/kg bw/d (being the highest dose tested). Again, the DRF study revealed severe effects at 300 mg/kg bw (mortality, reduced food intake, irritation in GI tract).

For C16-DMA the screening study according to OECD TG 422 revealed different NOAELs for male and female animals. For females the NOAEL is 150 mg/kg bw/d (highest dose tested) whereas for males the NOAEL is 100 mg/kg bw/d (dose levels: 15, 50, 100, 150 mg/kg bw/d) based on reduced body weight and weight gain in males at 150 mg/kg bw/d. In the corresponding DRF study a LOEL of 50 mg/kg bw/d for males and females was reported, based on for example changes in body weight and  food consumption, inflammatory markers and organ weights. A NOAEL is not provided for this study.

In the 14-day DRF study for C18-DMA a NOAEL of 50 mg/kg bw/d is reported for male and female rats. THE NOAEL is based on reduced body weight gain and food consumption, changes in organ weights and irritation in the GI tract. As outlined above, one OECD 408 study is planned either on C12-14 DMA or C16-DMA. The results will be included in IUCLID and this endpoint summary when available.

As outlined above, subchronic studies according to OECD TG 408 are planned for C12-14 DMA and C16-DMA. Currently no subchronic or chronic studies with members of the DMA-category are available. To fill this data gap, read-across to DMAOs is performed. In a chronic study with C10-16 DMAO (Cardin et al., 1985, reliability 2, equivalent to OECD guideline 453) decreased body weights at the highest dose tested (87.4 mg/kg bw/d for males and 107 mg/kg bw/d for females) was reported. No other adverse effects were observed, resulting in a NOAEL of 42.3 and 52.6 mg/kg bw/d for males and females, respectively. The animals were exposed for 2 years.

Other studies with repeated exposure to DMAOs are included as supporting evidence.

For oral exposure a reliable guideline study (equivalent to OECD 422, reliability 1) is available for C12-18-DMAO. In this study, male and female rats were exposed for at least 31 days (males) or longer (females: 14 days prior to mating and during the mating period, pregnancy and at least 3 days of lactation). The NOEL in this study was 40 mg/kg bw/d based on pathological changes in the forestomach and the mesenteric lymph nodes and signs of systemic toxicity (salivation) at dose levels of 100 and/or 200 mg/kg bw/d.

Lijinsky et al. (1984, reliability 3) reported a chronic study in rats. The animals were exposed for 93 weeks to ca. 250 mg C12-DMAO/kg bw/d. There was no effect on life span, no other data are available.

There are three additional studies with DMAOs. However, they were only available from a secondary source (OECD SIDS, 2006) which gives them a reliability of 4:

A 90-day toxicity study with C10-16-DMAO in rats determined a NOAEL of 80 mg/kg bw/d (females) and 63 mg/kg bw/d (males) (The Procter & Gamble, 1980). The NOAEL for females was based on decreases in mean body weight at 150 and 301 mg/kg bw/d (no other effects observed). Opthalmoscopic examination revealed lenticular opacities pertaining to the posterior cortex of the lens in males at 112 and 236 mg/kg bw/d. The body weight of the males was reduced in the highest dose group (236 mg/kg bw/d).

In a 32-week feeding study with C10-16-DMAO in rabbits, a NOAEL of 40 mg/kg bw/d (males) is established (The Procter & Gamble, 1977A). Decreased alkaline phosphatase levels and an increased liver/body weight ratio were noted in males at 196 mg/kg bw/d.

These NOAEL values were confirmed by the results of a two-generation study in rats (Lion Corporation, 1978B) which reported a NOAEL > 40 mg/kg bw/d (see section 5.9).

The toxicity patterns of all these studies are comparable to each other: The NOAEL and NOEL for systemic effects experimentally obtained is between 50 and 150 mg/kg in the available studies.  

 

For the chronic toxicity assessment of DMAs the use of 50 mg/kg as systemic NOAEL is considered to be sufficiently conservative, since there was no systemic effect present at this dose level. This approach is also in line with the approach taken by EFSA (2007), in which NOAEL of 50 mg/kg for DMAOs (based on Cardin et al, 1985) is used for the derivation of a TDI for DMAs in food contact materials.

 

Repeated-dose toxicity of DMAOs was also tested after dermal exposure. A reliable guideline study (Cardin et al, 1985, reliability 2, equivalent to OECD guideline 453) is available for C10-16-DMAO. In this study, mice were exposed for 2 years. The highest concentration tested (0.26%, corresponding to 5.6 mg/kg bw/d) did not result in any effects except for skin irritation (diffuse acanthosis and hyperkeratosis), which was observed microscopically in the high dose group of mice. Therefore, a NOAEL of ≥ 0.26% (5.6 mg/kg bw/d, highest dose tested) was established for systemic toxicity.

This result is supported by four non-reliable studies with DMAOs on rabbit skin (Petersen, 1988 a, b, c and Pang, 1994). Petersen exposed rabbits for 28 or 91 days against to 0, 0.3, 0.6 and 1.5 mg/kg bw/d or 0, 0.5, 1.0 and 2.5 mg/kg bw/d, administered as C12-14-DMAO. There were no signs of systemic toxicity or compound-related microscopic or macroscopic lesions in any dosage group. Therefore, the NOAEL values were 1.5 mg/kg bw/d and 2.5 mg/kg bw/d, the highest dosages tested. Pang exposed rabbits for 28 days to 0 or 6 mg/kg bw/d. Only slight irritation was observed, no significant changes in mean body weight, clinical observations, mean absolute organ weights or organ-to-body weight ratios were observed. Therefore, the NOEL for systemic effects was 6 mg/kg bw/d, the highest dose tested.

All in all, it can be stated that studies using DMAO as test substances are indicative of absence of systemic effects at skin irritation dose levels.

 

The overall NOAEL for repeated-dose toxicity after oral administration is 50 mg/kg bw/d. This is in agreement with the evaluation by EFSA (2007), which also identified a NOAEL of 50 mg/kg from the same study (Cardin et al, 1985) for the derivation of a TDI for DMAs in food contact materials. Unspecific toxic effects like effects on body weight or health conditions were observed at doses ≥ 100 mg/kg bw/day.

Data on dermal application indicate NOAEL values for DMAOs between 2 and 6 mg/kg bw/d, i.e. the highest doses tested. No specific systemic effects were observed at doses already causing local skin effects. Therefore, the members of the DMA category do not have to be classified for specific target organ toxicity – repeated exposure or repeated dose toxicity according to Regulation (EC) No 1272/2008

Justification for classification or non-classification

The NOAEL and NOEL for systemic effects experimentally obtained after oral exposure is between 50 and 150 mg/kg in the available studies. After dermal application of DMAOs no systemic toxicity was observed up to doses causing local effects on the skin (about 6 mg/kg bw/d).

Based on the category approach and the available data for repeated oral exposure, members of the DMA category do not have to be classified for specific target organ toxicity – repeated exposure or repeated dose toxicity according to Regulation (EC) No 1272/2008.