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Description of key information

Data from two key studies (one dermal subacute toxicity study and one oral subchronic toxicity study) are available. Furthermore there is additional information from an oral 14-day range finding study. There were no treatment related adverse effects in any of these studies up to the limit dose tested. In the dermal subacute toxicity study, there was in addition no evidence for local toxicity. From the results presented, a NOAEL of at least 1000 mg/kgbw/day was established for dermal subacute and oral subchronic toxicity to male and female rats.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 October 2011 - 19 January 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles.
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: - Japanese Chemical Substances Control Law 1987, Notification of Nov. 21 2003 by MHLW (No. 1121002), METI (No. 2) and ME (No. 031121002).
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Wistar strain, Crl:WI(Han)
Sex:
male/female
Details on test animals or test system and environmental conditions:
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Young adult animals (approx. 6 weeks old)
- Weight at study initiation: Body weight variation was within +/- 20% of the sex mean. (males: 156-176 grams; females: 93-123 grams)
- Housing: Group housing of 5 animals per cage in labeled Macrolon cages
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.9 – 23.4
- Humidity (%): 41 - 95
- Air changes (per hr): approx 15
- Photoperiod (hrs dark / hrs light): 12/12
Temporary deviations from the maximum level of relative humidity occurred. Laboratory historical data do not indicate an effect of the deviations.

IN-LIFE DATES: From: 17 October 2011 - 19 January 2012
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing, and were homogenized to visually acceptable levels. Adjustment was made for density and purity of the test substance.

VEHICLE:
Water

DOSE VOLUME:
5 ml/kg body weight. Actual dose volumes were calculated according to the latest body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted during the treatment phase, according to a validated method (NOTOX project 454117). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations; in weeks 1, 6 and 13). The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration for solutions. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.

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 90% and 110%).

A small response at the retention time of the test substance was observed in the chromatograms of the Group 1 formulation prepared for use in Week 6. This was considered to have been introduced during sample pretreatment, since the response was not observed in the duplicate test sample. In all other formulations of Group 1, no test substance was detected.

The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).
Duration of treatment / exposure:
At least 90 days.
Frequency of treatment:
Once daily, 7 d/w.
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dose levels were based on results of a 14-day oral range finding study with PC-2011-350 by daily gavage in the rat (NOTOX project 497623).
Positive control:
No.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: yes
- Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS:yes
- Time schedule: At least once daily from start of treatment onwards, detailed clinical observations were made in all animals after dosing. Once prior to start of treatment and at weekly intervals during the treatment phase pre-dose clinical observations were performed outside the home cage in a standard arena. The time of onset, grade and duration of any observed signs was recorded.
Arena observations were conducted prior to dosing instead of after dosing. No peak effect of occurrence of clinical signs was noted during the dose range finding study. Therefore, the timing of conduct of the arena observations was considered not critical.

BODY WEIGHT: yes
- Time schedule for examinations: Weekly.

FOOD EFFICIENCY: yes
- 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
Food consumption was measured off-line on Day 9 instead of Day 8. Based on conducted measurements, an adequate assessment of the food intake could be made. A correction was made afterwards in the calculated food intake data to account for the difference in measurement period of food intake during the first two weeks.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: yes
- Time schedule for examinations: at pretest: all animals (including spare animals), at week 13: Groups 1 and 4.

HAEMATOLOGY: yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: yes
- How many animals: all animals
- Parameters checked: According to test guidelines

CLINICAL CHEMISTRY: yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: yes
- How many animals: all animals
- Parameters checked: According to test guidelines

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: yes
- Time schedule for examinations: During week 12-13 of treatment
- Dose groups that were examined: all animals of Groups 1 and 4
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex and grip strength, motor activity test.
Sacrifice and pathology:
GROSS PATHOLOGY: yes
- All animals were fasted overnight with a maximum of 20 hours prior to planned necropsy, but water was provided. Animals surviving to scheduled necropsy were deeply anaesthetised and subsequently exsanguinated.
- Dose groups that were examined: all groups
- Tissues/organs checked: According to test guidelines

ORGAN WEIGHTS: yes
Organs checked according to test guidelines

HISTOPATHOLOGY: yes
According to test guidelines
For one animal of Group 4 one mandibular lymph node was available for histopathology. Sufficient data was available for histopathological evaluation.
Statistics:
- 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.
Motor activity data was subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences followed by the Wilcoxon test to compare the treated groups to the control group.
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
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:
no effects observed
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
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:
MORTALITY AND CLINICAL SIGNS
No mortality occurred during the study period.
No clinical signs of toxicity were noted during the observation period. Incidental findings that were noted included scabs, a broken tail apex, elongated teeth (indicated in the tables as “broken upper incisors”) and salivation. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological significance.

BODY WEIGHT AND WEIGHT GAIN
Body weights and body weight gain of treated animals remained in the same range as controls over the study period.

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

OPHTHALMOSCOPIC EXAMINATION
No toxicologically significant ophthalmology findings were noted. Incidental ophthalmology findings at pretest or in week 13 consisted of pinpoint or multifocal corneal opacities, central lens opacity, persistent hyaloid vessel remnants, posterior cataract due to hyaloid remnants, and focal corneal oedema. The nature and incidence of these findings was within the range considered to be normal for rats of this age and strain, and therefore considered to be of no toxicological relevance.

HAEMATOLOGY
No toxicologically relevant changes occurred in haematological parameters of treated rats. Any statistically significant changes in haematological parameters were considered to be of no toxicological significance as they occurred in the absence of a (clear) dose-related trend and remained within the range considered normal for rats of this age and strain. These changes consisted of lower white blood cell (WBC) red blood cell counts in males at 1000 mg/kg, higher mean corpuscular haemoglobin (MCH) in males and females at 1000 mg/kg, higher mean corpuscular volume (MCV) in males at 100 and 300 mg/kg, lower reticulocyte counts in females at 300 mg/kg, lower haematocrit level in females at 100 mg/kg, higher mean corpuscular haemoglobin concentration (MCHC) in females at 100, 300 and 1000 mg/kg, and higher platelet counts in females at 300 and 1000 mg/kg.

CLINICAL CHEMISTRY
No toxicologically relevant changes occurred in clinical biochemistry parameters of treated rats. Any statistically significant changes in clinical biochemistry parameters were considered to be of no toxicological significance as they occurred in the absence of a (clear) dose-related trend and remained within the range considered normal for rats of this age and strain. These changes consisted of lower sodium and chloride levels in males at 100 mg/kg, higher total bilirubin and urea levels in females at 1000 mg/kg, higher creatinine levels in females at 100, 300 and 1000 mg/kg, and lower potassium levels in females at 100 mg/kg.

NEUROBEHAVIOUR
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals of the control group and at 1000 mg/kg. Motor activity was similar between the 1000 mg/kg and control group. Both groups showed a similar motor activity habituation profile with high activity in the first interval that decreased over the duration of the test period.

ORGAN WEIGHTS
No toxicologically significant changes were noted in organ weights and organ to body weight ratios. Statistically significant changes in organ weights were considered not to be a sign of toxicity as they occurred in the absence of a (clear) dose-related trend and remained within the range considered normal for rats of this age and strain. These changes consisted of lower absolute and relative spleen and prostate weight in males at 100 mg/kg, lower absolute and relative seminal vesicle weight in males at 100 and 300 mg/kg, higher absolute brain weight in females at 300 mg/kg, and lower absolute and relative spleen weight in female at 300 mg/kg.

GROSS PATHOLOGY
Necropsy did not reveal any toxicologically relevant alterations. The incidence of macroscopic findings among control and treated animals was within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related incidence trend. These necropsy findings were therefore considered to be of no toxicological relevance, and included red foci on the lungs, an accessory liver on the caudate lobe, pelvic dilation of the kidneys, flaccid seminal vesicles, a tan focus on the preputial or clitoral glands, tan discolouration of the clitoral glands, enlarged spleen with irregular surface, a bent tail bone, a yellowish hard or soft nodule on the epididymal adipose tissue, fluid in the uterus, red discolouration of the mandibular lymph nodes and alopecia.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no microscopic findings recorded which could be attributed to treatment with the test substance. All microscopic findings were within the range of background pathology encountered in Wistar rats of this age and strain and occurred at similar incidences and severity in both control and treated rats.
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: No toxicity was observed up to the highest dose level tested.
Critical effects observed:
not specified
Conclusions:
From the results presented in this report a No Observed Adverse Effect Level (NOAEL) of at least 1000 mg/kg was established.
Executive summary:

In a subchronic toxicity study (according to OECD Guideline 408), the testsubstance was administered to 10 Wistar rats/sex/dose by gavage at dose levels of 0, 100, 300 and 1000 mg/kg for 90 consecutive days.

There were no compound related effects in mortality, clinical signs, functional observations, ophthalmoscopy, body weight and weight gain, food consumption and food efficiency, hematology, clinical chemistry, organ weights, gross and histologic pathology or neurobehaviour.

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

This subchronic toxicity study in the rat is acceptable and satisfies the guideline requirements for a OECD 408 study in rats.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Two studies are available (one 14-day dose range finding study and one oral subchronic toxicity study). The available studies are GLP compliant guideline studies with reliability 1. The overall quality of the database is therefore high.

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.9 (Repeated Dose (28 Days) Toxicity (Dermal))
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
Species : Rat: Wistar Crl:(WI) BR (outbred, SPF-Quality), Recognised by international guidelines as the recommended test system (e,g, OECD, EC).
Source : Charles River Deutschland, Sulzfeld, Germany.
Age and body weight : At commencement of the main study animals were approximately 10 weeks old, and weight ranges were as follows: Males: 338 - 380 grams; females: 190 - 220 grams, These weight ranges provided animals of a size which facilitated the conduct of the test.
Number of animals : 20 males, 20 females (females were nulliparous and non-pregnant).
Randomisation : Prior to commencement of treatment, by computer-generated random algorithm according to body weight, with all animals within ±20% of the sex mean.
Identification : Tattoo and earmark,
Health inspection : A health inspection was performed prior to commencement of treatment to ensure that the animals were in a good state of health,special attention was paid to the skin to be treated, which was intact and free from abnormalities.
Conditions : Animals were housed in a controlled environment, in which optimal conditions were considered to be approximately 15 air changes per hour, a temperature of 21 ,0 ± 3,0°C (actual range: 19,8 - 22,1 °C), a relative humidity of 30-70% (actual range: 40 - 69%) and 12 hours artificialfluorescent light and 12 hours darkness per day. Temporary fluctuations from the IighUdark cycle (with a maximum of 1 hour) occurred due to
performance of functional observations in the room, Based on laboratory historical data, these fluctuations were considered not to have affected the study integrity.
Accommodation : The acclimatisation period was at least 5 days before the start of treatment under laboratory conditions. During the acclimatisation and treatment phase, animals were housed individually in labelled polycarbonate cages (Mill type; height 18 cm). During overnight activity monitoring, animals were housed individually in Macrolon plastic cages (Mill type; height 15 cm,). Sterilised sawdust was provided as bedding material (Woody-Clean type 3/4, Tecnilab-BMI BV, Someren, The Netherlands) and paper as cage-enrichment (Enviro-dri, BMI, Helmond, The Netherlands), No cage-enrichment was provided during overnight activity monitoring. Results of bedding analyses for contaminants are examined and archived.
Diet : Free access to standard pelleted laboratory animal diet (from Altromin (code VRF 1), Lage, Germany)
Water : Free access to tap water. Certificates of analysis (performed quarterly) were examined and archived.
Results of analysis for ingredients and/or contaminants of diet, bedding, paper and water were assessed and did not reveal any findings that were considered to have affected study integrity.
Type of coverage:
occlusive
Vehicle:
water
Details on exposure:
Method : Dermal application. Formulations were placed on a magnetic stirrer during dosing.
Clipping : 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.
Application : The test substance was applied in an area of approx. 10% of the total body surface, i.e. approx. 25 cm2 for males and 18 cm2 for females. The test substance formulation was held in contact with the skin with a dressing, consisting of a surgical gauze patch (Surgy 1 D)*, successively covered with aluminium foil and Coban* flexible bandage. A piece of Micropore tape* was additionally used for fixation of the bandages in females only.
*. Manufacturers: Laboratoires Stella s.a., Liege, Belgium (surgical gauze) and 3M,
Frequency : Once daily for at least 28 days, 7 days per week. Application was performed approximately the same time each day. Animals were treated up to the day prior to necropsy.Sf. Paul, Minnesota, U.S.A (Caban & Micropore).
Exposure period : 6 hours, after which the dressing was removed and the skin cleaned of residual test substance using water. Inadvertently, the bandage from animal no. 27 was not removed on day 10. During the exposure period the dressing was checked regularly, at least every 2 hours (ie. after application, and approximately 1, 3 and 5 hours after application). In case of removal of the bandage before the 5-hour observation time point, a new dressing with fresh test substance was applied.
Dose volume : The test substance was dosed at 5 ml/kg b.w. 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:
Analytical method : Quantitative analysis was based on the test sUbstance peak in the HPLC chromatograms (see NOTOX project 454117: "Development and validation of an analytical method for the analysis of FORMAMIDOPROPYLBETAINE in Milli-U water").
Analytical conditions:
Column:
Stationary phase : YMC-Pack ODS-AQ
Dimensions : 250 mm x 4.6 mm id.; dp = 5 µm
Brand : YMC, Kyoto, Japan
Mobile phase : 0.05% TFA in Milli-Q water
Flow : 1.0 ml/min
Injection volume : 20 µL
Detection : UV absorption at a wavelength of 205 nm
Duration of treatment / exposure:
6 hours per day for 28 days
Frequency of treatment:
once daily for 28 days
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg b.w.
Basis:
other: nominal doses, analytically verified
No. of animals per sex per dose:
5
Control animals:
yes
Details on study design:
- Dose selection rationale: based on the results of a 5-day range finding study
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: no satellite groups
- Post-exposure recovery period in satellite groups: --
- Section schedule rationale (if not random): random
Observations and examinations performed and frequency:
Mortality / Viability : At least twice daily.
Clinical signs : At least once daily from day 1 onwards, immediately after the exposure period. The symptoms were graded according to fixed scales and the time of onset, degree and duration were recorded: Maximum grade 1: grade 0 = absent, grade 1 = present Maximum grade 3 or 4: grade 1 = slight, grade 2 = moderate, grade 3 = severe, grade 4 = very severe.
Functional Observations : During week 4 of treatment, the following tests were performed on all animals after removal of the bandage (abbreviations mentioned in the respective tables indicated between brackets): hearing ability (HEARING), pupillary reflex (PUPIL LlR), static righting reflex (STATIC R) and grip strength (GRIP) (Score 0 = normal/present, score 1 = abnormal/absent). Motor activity test (recording period: 12 hours during overnight for individual animals, using a computerised monitoring system, Pearson Technical Services, Debenham, Stowmarket, England).
Body weights : Prior to dosing on days 1, 8, 15, 22 and 28.
Food consumption : Weekly.
Water consumption : Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.
Clinical Laboratory Investigations : Blood samples were collected under iso-flurane anaesthesia (Abbott Laboratories Ltd., Zwolle, The Netherlands) immediately prior to scheduled post mortem examination, between 7.30 and 10.30 a.m. The animals were fasted overnight (with a maximum of 20 hours) before blood sampling, but water was provided. Blood samples were drawn from the retro-orbital sinus of all rats/sex/group and collected into tubes (Greiner Bio-One, Bad Haller, Austria) prepared with EDTA for haematological parameters (0.5 ml), with citrate for clotting tests (0.9 ml) and Liheparin treated tubes for clinical biochemistry parameters (0.5 ml). The following parameters were determined: Haematology, Clotting Potential, Clinical Biochemistry.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. All animals surviving to the end of the observation period were deeply anaesthetised using isoflurane vapour (Abbott Laboratories Ltd., Zwolle, The Netherlands) and subsequently exsanguinated. All animals assigned to the study were necropsied and descriptions of all
macroscopic abnormalities recorded.
HISTOPATHOLOGY: Yes (see table)
Other examinations:
The following organ weights (and terminal body weight) were recorded from the animals on the scheduled day of necropsy:
Adrenals, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Spleen, Testes, Thymus, Uterus.
Statistics:
The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test1 (many-to-one t-test) based on a pooled variance estimate wasapplied for the comparison of the
treated groups and the control groups for each sex.
- The Steel-tese (many-to-one rank test) was applied when the data could not be assumed to follow a normal distribution.
- The exact Fisher-tese 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. No statistical analysis was performed on histopathology findings. 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:
no effects observed
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 examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
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:
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 : No mortality occurred during the study period. No clinical signs of toxicity were observed throughout the treatment period. General) hyperthermia or hyperthermia of the treated skin of some females at 300 and 1000 mg/kg/day was not correlated to any (local) morphological abnormalities, but corresponded to observations in the pilot study. This finding was also not consistently seen with continuing treatment. Yellow urine noted among the females at 1000 mg/kg/day was only observed during week 2 of treatment and had no morphological or clinical pathology correlates. Clonic spasms were incidentally shown by two females only (animal nos. 21 and 30) during/after application of the bandage in week 3/4. The occurrence/incidence of this finding showed no relationship to the dose. These findings were therefore considered to be of no toxicological relevance. At 100 mg/kg/day and higher, scabs, scales and/or focal erythema of the treated skin were observed, and additionally at 300 mg/kg/day a wound and scabs on the chest or scabs on the abdomen in females were noted during the treatment period. The incidence of these findings did not show a relationship to the dose. Therefore, the treatment procedure rather than local effects of the test substance were considered to have caused these signs. Incidental findings that were noted included chromodacryorrhoea, watery discharge from the eyes, hypothermia, alopecia and diarrhoea. These findings are occasionally noted in rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological significance.

BODY WEIGHT AND WEIGHT GAIN : Body weights and body weight gain of treated animals remained in the same range as controls over the 4-week study period. Among the dose groups slight weight loss was observed among most males and some females at the end of the treatment period, as well as in one male at 100 and 300 mg/kg/day respectively. The incidence of slight weight loss showed no relationship to the dose, and was considered to be due to the burden of dermal treatment.

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

FOOD EFFICIENCY : not examined

WATER CONSUMPTION : not affected

OPHTHALMOSCOPIC EXAMINATION : not examined

HAEMATOLOGY : No toxicologically relevant changes occurred in haematological parameters of treated rats. The statistically significant lower red blood cell counts, haemoglobin and haematocrit levels in males at 100, 300 and/or 1000 mg/kg/day showed no dose-response relationship. Also, the
mean control red blood cell level was slightly high, and any supportive changes indicative of an effect on red blood cell turnover were absent. These changes were therefore considered to be of no toxicological significance.

CLINICAL CHEMISTRY : No toxicologically relevant changes occurred in clinical biochemistry parameters of treated rats. The statistically significant higher glucose level of males at 1000 mg/kg/day was not supported by any (morphological) changes indicative of organ dysfunction, and the change was of a slight nature. Values in males at 100 and/or 300 mg/kg/day achieving a level of statistical significance when compared to controls occurred in the absence of a treatment-related distribution. These changes included lower total protein, albumin and calcium levels. No toxicological significance was ascribed to these changes.

NEUROBEHAVIOUR : Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals. The variation in motor activity was similar between treated and control groups and did not indicate a relation with treatment

ORGAN WEIGHTS : Organ weights and organ to body weight ratios of treated animals were considered to be similar to those of control animals.

GROSS PATHOLOGY : Necropsy did not reveal any toxicologically relevant alterations. Liver abnormalities noted in male nos. 10, 14 and 20 (100, 300 and 1000 mg/kg/day, respectively) correlated microscopically to liver torsion/infarction and were considered to be due to the treatment procedure. Other incidental findings among control and treated animals included a reduced size of the seminal vesicles, red foci on the thymus, enlarged mandibular lymph node, red discolouration of the mandibular lymph node, scab formation on the treated skin, and fluid in the uterus. These findings are occasionally seen among rats used in these types of studies. In the absence of a treatment-related distribution they were considered changes of no toxicological significance.

HISTOPATHOLOGY: NON-NEOPLASTIC. There were no microscopic findings recorded which could be attributed to treatment with the test substance. Lobar torsion/infarction of the liver was recorded in male nos. 10, 14 and 20 (100, 300 and 1000 mg/kg/day, respectively) and correlated to the necropsy findings in the liver. This finding is considered to result from the animals rotating in the cage in an effort to dislodge the bandage and is a typical non-specific finding in dermal studies utilizing bandaging. Slight degrees of ulcerative dermal inflammation were recorded in the treated skin of two males at 100 mg/kg/day and one female at 1000 mg/kg/day. In view of the minor degree of this finding and the lack of a dose response it was considered to be a non-specific response rather than an indicator of an irritant potential of the test substance. All other microscopic findings were within the range of background pathology encountered in Wistar rats of this age and strain and occurred at similar incidences and severity in both control and treated rats.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: nominal doses analytically verified
Critical effects observed:
not specified

Body weight development [g]

Treatment

 

Group 1 Control

Group2    100 mg/kg

Group3    300 mg/kg

Group4    1000 mg/kg

Males

Day 1

Mean

359

355

363

363

Week 1

ST. DEV.

12.6

12.5

13.1

10.0

 

N

5

5

5

5

Day 8

Mean

369

360

371

376

Week 2

ST. DEV.

11.1

13.4

10.4

11.6

 

N

5

5

5

5

Day 15

Mean

390

384

393

396

Week 3

ST. DEV.

16.8

13.5

11.9

13.3

 

N

5

5

5

5

Day 22

Mean

402

402

410

412

Week 4

ST. DEV.

18.5

14.1

7.8

22.7

 

N

5

5

5

5

Day 28

Mean

396

398

406

411

Week 4

ST. DEV.

18.7

10.4

7.9

19.7

 

N

5

5

5

5

Females

Day 1

Mean

204

203

200

205

Week 1

ST. DEV.

6.5

11.2

3.7

8.4

 

N

5

5

5

5

Day 8

Mean

214

212

207

216

Week 2

ST. DEV.

7.3

7.9

9.6

6.9

 

N

5

5

5

5

Day 15

Mean

225

226

231

231

Week 3

ST. DEV.

9.9

16.7

9.6

16.0

 

N

5

5

5

5

Day 22

Mean

239

241

242

245

Week 4

ST. DEV.

9.9

16.7

9.6

16.0

 

N

5

5

5

5

Day 28

Mean

240

244

241

245

Week 4

ST. DEV.

14.1

16.1

13.5

12.6

 

N

5

5

5

5

 

* / **Dunnett-Test based on pooled variance significant at 5% (*) or 1% (**) level

 

Conclusions:
Wistar rats were treated with FORMAMIDOPROPYLBETAINE for 28 consecutive days by dermal application at dose levels up to 1000 mg/kg/day. There was no evidence of systemic (or local) toxicity in response to treatment with the test substance.
There were no 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 that were considered to be of toxicological significance.
In the dermal repeated dose toxicity study the NOAEL is >= 1000 mg/kg bw/day for male and female rats. A point of departure for the assessment of systemic toxicity after dermal exposure could not be derived due to the lack of substance related critical health effects up to and including the limit dose of the study design. As an actual value for the NOAEL could not be determined due to limit dose testing in this study type, it could be much higher than 1000 mg/kg bw/day. For risk assessment purposes a NOAEL of 1000 mg/kg/day (nominal dose) was established.
Executive summary:

Repeated dose (28-days) dermal toxicity study with FORMAMIDOPROPYLBETAINE by daily exposure in the rat.

The study was based on the following guidelines: EEC Directive 92/69/EEC, B.9: "Repeated Dose (28 days) Toxicity (dermal)". 1992 and OECD 410, "Repeated Dose Dermal Toxicity: 21/28-day Study", 1981.

Based on the results of a 5-day range finding study, the dose levels for the 28-day toxicity study were selected to be 0,100,300 and 1000 mg/kg/day. The test substance was topically applied for approximately 6 hours/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. Chemical analyses of formulations were conducted once during the study to assess accuracy and homogeneity. 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 and homogeneity of formulations of test substance in Milli-U water were demonstrated by analyses. There was no evidence of systemic (or local) toxicity in response to treatment with the test substance.

Conclusion

In the dermal repeated dose toxicity study the NOAEL is >= 1000 mg/kg bw/day for male and female rats. A point of departure for the assessment of systemic toxicity after dermal exposure could not be derived due to the lack of substance related critical health effects up to and including the limit dose of the study design. As an actual value for the NOAEL could not be determined due to limit dose testing in this study type, it could be much higher than 1000 mg/kg bw/day. For risk assessment purposes a NOAEL of 1000 mg/kg/day (nominal dose) was established.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Data from a GLP compliant guideline study with reliability 1.

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.9 (Repeated Dose (28 Days) Toxicity (Dermal))
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
Species : Rat: Wistar Crl:(WI) BR (outbred, SPF-Quality), Recognised by international guidelines as the recommended test system (e,g, OECD, EC).
Source : Charles River Deutschland, Sulzfeld, Germany.
Age and body weight : At commencement of the main study animals were approximately 10 weeks old, and weight ranges were as follows: Males: 338 - 380 grams; females: 190 - 220 grams, These weight ranges provided animals of a size which facilitated the conduct of the test.
Number of animals : 20 males, 20 females (females were nulliparous and non-pregnant).
Randomisation : Prior to commencement of treatment, by computer-generated random algorithm according to body weight, with all animals within ±20% of the sex mean.
Identification : Tattoo and earmark,
Health inspection : A health inspection was performed prior to commencement of treatment to ensure that the animals were in a good state of health,special attention was paid to the skin to be treated, which was intact and free from abnormalities.
Conditions : Animals were housed in a controlled environment, in which optimal conditions were considered to be approximately 15 air changes per hour, a temperature of 21 ,0 ± 3,0°C (actual range: 19,8 - 22,1 °C), a relative humidity of 30-70% (actual range: 40 - 69%) and 12 hours artificialfluorescent light and 12 hours darkness per day. Temporary fluctuations from the IighUdark cycle (with a maximum of 1 hour) occurred due to
performance of functional observations in the room, Based on laboratory historical data, these fluctuations were considered not to have affected the study integrity.
Accommodation : The acclimatisation period was at least 5 days before the start of treatment under laboratory conditions. During the acclimatisation and treatment phase, animals were housed individually in labelled polycarbonate cages (Mill type; height 18 cm). During overnight activity monitoring, animals were housed individually in Macrolon plastic cages (Mill type; height 15 cm,). Sterilised sawdust was provided as bedding material (Woody-Clean type 3/4, Tecnilab-BMI BV, Someren, The Netherlands) and paper as cage-enrichment (Enviro-dri, BMI, Helmond, The Netherlands), No cage-enrichment was provided during overnight activity monitoring. Results of bedding analyses for contaminants are examined and archived.
Diet : Free access to standard pelleted laboratory animal diet (from Altromin (code VRF 1), Lage, Germany)
Water : Free access to tap water. Certificates of analysis (performed quarterly) were examined and archived.
Results of analysis for ingredients and/or contaminants of diet, bedding, paper and water were assessed and did not reveal any findings that were considered to have affected study integrity.
Type of coverage:
occlusive
Vehicle:
water
Details on exposure:
Method : Dermal application. Formulations were placed on a magnetic stirrer during dosing.
Clipping : 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.
Application : The test substance was applied in an area of approx. 10% of the total body surface, i.e. approx. 25 cm2 for males and 18 cm2 for females. The test substance formulation was held in contact with the skin with a dressing, consisting of a surgical gauze patch (Surgy 1 D)*, successively covered with aluminium foil and Coban* flexible bandage. A piece of Micropore tape* was additionally used for fixation of the bandages in females only.
*. Manufacturers: Laboratoires Stella s.a., Liege, Belgium (surgical gauze) and 3M,
Frequency : Once daily for at least 28 days, 7 days per week. Application was performed approximately the same time each day. Animals were treated up to the day prior to necropsy.Sf. Paul, Minnesota, U.S.A (Caban & Micropore).
Exposure period : 6 hours, after which the dressing was removed and the skin cleaned of residual test substance using water. Inadvertently, the bandage from animal no. 27 was not removed on day 10. During the exposure period the dressing was checked regularly, at least every 2 hours (ie. after application, and approximately 1, 3 and 5 hours after application). In case of removal of the bandage before the 5-hour observation time point, a new dressing with fresh test substance was applied.
Dose volume : The test substance was dosed at 5 ml/kg b.w. 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:
Analytical method : Quantitative analysis was based on the test sUbstance peak in the HPLC chromatograms (see NOTOX project 454117: "Development and validation of an analytical method for the analysis of FORMAMIDOPROPYLBETAINE in Milli-U water").
Analytical conditions:
Column:
Stationary phase : YMC-Pack ODS-AQ
Dimensions : 250 mm x 4.6 mm id.; dp = 5 µm
Brand : YMC, Kyoto, Japan
Mobile phase : 0.05% TFA in Milli-Q water
Flow : 1.0 ml/min
Injection volume : 20 µL
Detection : UV absorption at a wavelength of 205 nm
Duration of treatment / exposure:
6 hours per day for 28 days
Frequency of treatment:
once daily for 28 days
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg b.w.
Basis:
other: nominal doses, analytically verified
No. of animals per sex per dose:
5
Control animals:
yes
Details on study design:
- Dose selection rationale: based on the results of a 5-day range finding study
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: no satellite groups
- Post-exposure recovery period in satellite groups: --
- Section schedule rationale (if not random): random
Observations and examinations performed and frequency:
Mortality / Viability : At least twice daily.
Clinical signs : At least once daily from day 1 onwards, immediately after the exposure period. The symptoms were graded according to fixed scales and the time of onset, degree and duration were recorded: Maximum grade 1: grade 0 = absent, grade 1 = present Maximum grade 3 or 4: grade 1 = slight, grade 2 = moderate, grade 3 = severe, grade 4 = very severe.
Functional Observations : During week 4 of treatment, the following tests were performed on all animals after removal of the bandage (abbreviations mentioned in the respective tables indicated between brackets): hearing ability (HEARING), pupillary reflex (PUPIL LlR), static righting reflex (STATIC R) and grip strength (GRIP) (Score 0 = normal/present, score 1 = abnormal/absent). Motor activity test (recording period: 12 hours during overnight for individual animals, using a computerised monitoring system, Pearson Technical Services, Debenham, Stowmarket, England).
Body weights : Prior to dosing on days 1, 8, 15, 22 and 28.
Food consumption : Weekly.
Water consumption : Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.
Clinical Laboratory Investigations : Blood samples were collected under iso-flurane anaesthesia (Abbott Laboratories Ltd., Zwolle, The Netherlands) immediately prior to scheduled post mortem examination, between 7.30 and 10.30 a.m. The animals were fasted overnight (with a maximum of 20 hours) before blood sampling, but water was provided. Blood samples were drawn from the retro-orbital sinus of all rats/sex/group and collected into tubes (Greiner Bio-One, Bad Haller, Austria) prepared with EDTA for haematological parameters (0.5 ml), with citrate for clotting tests (0.9 ml) and Liheparin treated tubes for clinical biochemistry parameters (0.5 ml). The following parameters were determined: Haematology, Clotting Potential, Clinical Biochemistry.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. All animals surviving to the end of the observation period were deeply anaesthetised using isoflurane vapour (Abbott Laboratories Ltd., Zwolle, The Netherlands) and subsequently exsanguinated. All animals assigned to the study were necropsied and descriptions of all
macroscopic abnormalities recorded.
HISTOPATHOLOGY: Yes (see table)
Other examinations:
The following organ weights (and terminal body weight) were recorded from the animals on the scheduled day of necropsy:
Adrenals, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Spleen, Testes, Thymus, Uterus.
Statistics:
The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test1 (many-to-one t-test) based on a pooled variance estimate wasapplied for the comparison of the
treated groups and the control groups for each sex.
- The Steel-tese (many-to-one rank test) was applied when the data could not be assumed to follow a normal distribution.
- The exact Fisher-tese 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. No statistical analysis was performed on histopathology findings. 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:
no effects observed
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 examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
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:
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 : No mortality occurred during the study period. No clinical signs of toxicity were observed throughout the treatment period. General) hyperthermia or hyperthermia of the treated skin of some females at 300 and 1000 mg/kg/day was not correlated to any (local) morphological abnormalities, but corresponded to observations in the pilot study. This finding was also not consistently seen with continuing treatment. Yellow urine noted among the females at 1000 mg/kg/day was only observed during week 2 of treatment and had no morphological or clinical pathology correlates. Clonic spasms were incidentally shown by two females only (animal nos. 21 and 30) during/after application of the bandage in week 3/4. The occurrence/incidence of this finding showed no relationship to the dose. These findings were therefore considered to be of no toxicological relevance. At 100 mg/kg/day and higher, scabs, scales and/or focal erythema of the treated skin were observed, and additionally at 300 mg/kg/day a wound and scabs on the chest or scabs on the abdomen in females were noted during the treatment period. The incidence of these findings did not show a relationship to the dose. Therefore, the treatment procedure rather than local effects of the test substance were considered to have caused these signs. Incidental findings that were noted included chromodacryorrhoea, watery discharge from the eyes, hypothermia, alopecia and diarrhoea. These findings are occasionally noted in rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological significance.

BODY WEIGHT AND WEIGHT GAIN : Body weights and body weight gain of treated animals remained in the same range as controls over the 4-week study period. Among the dose groups slight weight loss was observed among most males and some females at the end of the treatment period, as well as in one male at 100 and 300 mg/kg/day respectively. The incidence of slight weight loss showed no relationship to the dose, and was considered to be due to the burden of dermal treatment.

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

FOOD EFFICIENCY : not examined

WATER CONSUMPTION : not affected

OPHTHALMOSCOPIC EXAMINATION : not examined

HAEMATOLOGY : No toxicologically relevant changes occurred in haematological parameters of treated rats. The statistically significant lower red blood cell counts, haemoglobin and haematocrit levels in males at 100, 300 and/or 1000 mg/kg/day showed no dose-response relationship. Also, the
mean control red blood cell level was slightly high, and any supportive changes indicative of an effect on red blood cell turnover were absent. These changes were therefore considered to be of no toxicological significance.

CLINICAL CHEMISTRY : No toxicologically relevant changes occurred in clinical biochemistry parameters of treated rats. The statistically significant higher glucose level of males at 1000 mg/kg/day was not supported by any (morphological) changes indicative of organ dysfunction, and the change was of a slight nature. Values in males at 100 and/or 300 mg/kg/day achieving a level of statistical significance when compared to controls occurred in the absence of a treatment-related distribution. These changes included lower total protein, albumin and calcium levels. No toxicological significance was ascribed to these changes.

NEUROBEHAVIOUR : Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals. The variation in motor activity was similar between treated and control groups and did not indicate a relation with treatment

ORGAN WEIGHTS : Organ weights and organ to body weight ratios of treated animals were considered to be similar to those of control animals.

GROSS PATHOLOGY : Necropsy did not reveal any toxicologically relevant alterations. Liver abnormalities noted in male nos. 10, 14 and 20 (100, 300 and 1000 mg/kg/day, respectively) correlated microscopically to liver torsion/infarction and were considered to be due to the treatment procedure. Other incidental findings among control and treated animals included a reduced size of the seminal vesicles, red foci on the thymus, enlarged mandibular lymph node, red discolouration of the mandibular lymph node, scab formation on the treated skin, and fluid in the uterus. These findings are occasionally seen among rats used in these types of studies. In the absence of a treatment-related distribution they were considered changes of no toxicological significance.

HISTOPATHOLOGY: NON-NEOPLASTIC. There were no microscopic findings recorded which could be attributed to treatment with the test substance. Lobar torsion/infarction of the liver was recorded in male nos. 10, 14 and 20 (100, 300 and 1000 mg/kg/day, respectively) and correlated to the necropsy findings in the liver. This finding is considered to result from the animals rotating in the cage in an effort to dislodge the bandage and is a typical non-specific finding in dermal studies utilizing bandaging. Slight degrees of ulcerative dermal inflammation were recorded in the treated skin of two males at 100 mg/kg/day and one female at 1000 mg/kg/day. In view of the minor degree of this finding and the lack of a dose response it was considered to be a non-specific response rather than an indicator of an irritant potential of the test substance. All other microscopic findings were within the range of background pathology encountered in Wistar rats of this age and strain and occurred at similar incidences and severity in both control and treated rats.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: nominal doses analytically verified
Critical effects observed:
not specified

Body weight development [g]

Treatment

 

Group 1 Control

Group2    100 mg/kg

Group3    300 mg/kg

Group4    1000 mg/kg

Males

Day 1

Mean

359

355

363

363

Week 1

ST. DEV.

12.6

12.5

13.1

10.0

 

N

5

5

5

5

Day 8

Mean

369

360

371

376

Week 2

ST. DEV.

11.1

13.4

10.4

11.6

 

N

5

5

5

5

Day 15

Mean

390

384

393

396

Week 3

ST. DEV.

16.8

13.5

11.9

13.3

 

N

5

5

5

5

Day 22

Mean

402

402

410

412

Week 4

ST. DEV.

18.5

14.1

7.8

22.7

 

N

5

5

5

5

Day 28

Mean

396

398

406

411

Week 4

ST. DEV.

18.7

10.4

7.9

19.7

 

N

5

5

5

5

Females

Day 1

Mean

204

203

200

205

Week 1

ST. DEV.

6.5

11.2

3.7

8.4

 

N

5

5

5

5

Day 8

Mean

214

212

207

216

Week 2

ST. DEV.

7.3

7.9

9.6

6.9

 

N

5

5

5

5

Day 15

Mean

225

226

231

231

Week 3

ST. DEV.

9.9

16.7

9.6

16.0

 

N

5

5

5

5

Day 22

Mean

239

241

242

245

Week 4

ST. DEV.

9.9

16.7

9.6

16.0

 

N

5

5

5

5

Day 28

Mean

240

244

241

245

Week 4

ST. DEV.

14.1

16.1

13.5

12.6

 

N

5

5

5

5

 

* / **Dunnett-Test based on pooled variance significant at 5% (*) or 1% (**) level

 

Conclusions:
Wistar rats were treated with FORMAMIDOPROPYLBETAINE for 28 consecutive days by dermal application at dose levels up to 1000 mg/kg/day. There was no evidence of systemic (or local) toxicity in response to treatment with the test substance.
There were no 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 that were considered to be of toxicological significance.
In the dermal repeated dose toxicity study the NOAEL is >= 1000 mg/kg bw/day for male and female rats. A point of departure for the assessment of systemic toxicity after dermal exposure could not be derived due to the lack of substance related critical health effects up to and including the limit dose of the study design. As an actual value for the NOAEL could not be determined due to limit dose testing in this study type, it could be much higher than 1000 mg/kg bw/day. For risk assessment purposes a NOAEL of 1000 mg/kg/day (nominal dose) was established.
Executive summary:

Repeated dose (28-days) dermal toxicity study with FORMAMIDOPROPYLBETAINE by daily exposure in the rat.

The study was based on the following guidelines: EEC Directive 92/69/EEC, B.9: "Repeated Dose (28 days) Toxicity (dermal)". 1992 and OECD 410, "Repeated Dose Dermal Toxicity: 21/28-day Study", 1981.

Based on the results of a 5-day range finding study, the dose levels for the 28-day toxicity study were selected to be 0,100,300 and 1000 mg/kg/day. The test substance was topically applied for approximately 6 hours/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. Chemical analyses of formulations were conducted once during the study to assess accuracy and homogeneity. 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 and homogeneity of formulations of test substance in Milli-U water were demonstrated by analyses. There was no evidence of systemic (or local) toxicity in response to treatment with the test substance.

Conclusion

In the dermal repeated dose toxicity study the NOAEL is >= 1000 mg/kg bw/day for male and female rats. A point of departure for the assessment of systemic toxicity after dermal exposure could not be derived due to the lack of substance related critical health effects up to and including the limit dose of the study design. As an actual value for the NOAEL could not be determined due to limit dose testing in this study type, it could be much higher than 1000 mg/kg bw/day. For risk assessment purposes a NOAEL of 1000 mg/kg/day (nominal dose) was established.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Quality of whole database:
Data from a GLP compliant guideline study with reliability 1.

Additional information

Subacute dermal toxicity:

Repeated dose (28-days) dermal toxicity study with Formamidopropyldimethylbetain by daily exposure in the rat.

The study was based on the following guidelines: EEC Directive 92/69/EEC, B.9: "Repeated Dose (28 days) Toxicity (dermal) ". 1992 and OECD 410, "Repeated Dose Dermal Toxicity: 21/28-day Study", 1981.

Based on the results of a 5-day range finding study, the dose levels for the 28-day toxicity study were selected to be 0,100,300 and 1000 mg/kg/day. The test substance was topically applied for approximately 6 hours/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. Chemical analyses of formulations were conducted once during the study to assess accuracy and homogeneity. 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

There were no 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 that were considered to be of toxicological significance. There was no evidence of systemic (or local) toxicity in response to treatment with the test substance.

Accuracy and homogeneity of formulations of test substance in Milli-U water were demonstrated by analyses. There was no evidence of systemic (or local) toxicity in response to treatment with the test substance.

Conclusion

In the dermal repeated dose toxicity study the NOAEL is >= 1000 mg/kg bw/day for male and female rats. A point of departure for the assessment of systemic toxicity after dermal exposure could not be derived due to the lack of substance related critical health effects up to and including the limit dose of the study design. As an actual value for the NOAEL could not be determined due to limit dose testing in this study type, it could be much higher than 1000 mg/kg bw/day. For risk assessment purposes a NOAEL of 1000 mg/kg/day (nominal dose) was established

Subchronic oral tocxicity

The substance was subjected to a compliance check by ECHA. It was decided (decision number: CCH-D-0000001299-68-04/F) that no screening test on reproductive toxicity has to be carried out, but a developmental toxicity study and a 90 day repeated dose toxicity study must be carried out.

In a subchronic toxicity study (according to OECD Guideline 408), the testsubstance was administered to 10 Wistar rats/sex/dose by gavage at dose levels of 0, 100, 300 and 1000 mg/kg for 90 consecutive days.

Results:

Any statistically significant changes in haematological parameters, clinical biochemistry parameters or organ weights were considered to be of no toxicological significance as they occurred in the absence of a (clear) dose-related trend and remained within the range considered normal for rats of this age and strain.

There were no compound related effects in mortality, clinical signs, functional observations, ophthalmoscopy, body weight and weight gain, food consumption and food efficiency, hematology, clinical chemistry, organ weights, gross and histologic pathology or neurobehaviour.

Conclusion:

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

As an actual value for the NOAEL could not be determined due to limit dose testing in this study type, it could be much higher than 1000 mg/kg bw/day. For risk assessment purposes a NOAEL of 1000 mg/kg/day was established


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Data from a GLP compliant guideline study with reliability 1.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
Data from a GLP compliant guideline study with reliability 1.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
Data from a GLP compliant guideline study with reliability 1.

Justification for classification or non-classification

In conclusion, the result of the available study indicates that Formamidopropyldimethylbetain, needs not be classified for repeated dose toxicity according to Directive 67/548/EEC as well as GHS Regulation EC No 1272/2008 and therefore labelling is not necessary.