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Description of key information

Subacute studies:



  • Rats NOAEL (gavage) = 150 mg/kg bw/day (basis: highest dose level tested) (BG Chemie, 1998, Key, Rel.1)

  • Rats NOEL (gavage) = 50 mg/kg bw/day (basis: miosis at 100 mg/kg bw/day in both sexes) (MHLW, 1997, K, rel.2)


Sub-chronic study:



  • Rats NOAEL (gavage) = 75 mg/kg bw/day (basis: mortality and generalized tremors at 150 mg/kg bw/day) (Charles River Laboratories, 2023, Key, Rel.1)

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Detailed study summary and tables available (main text in japanese)
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
other: Guidelines for 28-day Repeat Dose Toxicity testing of Chemicals (Japan)
Deviations:
yes
Remarks:
- recovery groups
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
no details given
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
male and female rats were given the different amountsdiluted in conr oil by gavage once daily over a period of 28 days
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
0, 50, 100, 200, 400 mg/kg bw/day
Basis:

No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
male and female rats were fed with 0, 50, 100, 200, 400 mg7kg bw/day over a period of 28 days,
rats dosed with 50 and 100 mg/kg bw/day were killed immediately after cessation of treatment:
groups of rats dosed with 200 and 400 mg/kg bw/day were aloud to recover for 14 days and then examined accordingly
Positive control:
not required
Observations and examinations performed and frequency:
clinical observation once a day
mortality body weight blood examinations hematology
clinical biochemistry, urinalysis
Sacrifice and pathology:
gross pathology, organ weight.microscopic examination:
brain, liver, kidney, spleen, adrenals,ovaries, testes, thymus,
respiratory system, integumentary system, cardiovascular system, reproductive system
hematopoietic system, urinary system, digestive system
Other examinations:
no data
Statistics:
Non-parametric analysis
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
mortality from week 2 at 400 mg/kg bw/d: males: 4/5; females.5/5
no control animal died
gross pathological examination revealed no obvious changes
histopathological examination:
inclusion bodies in hepatocytes (m+f); degeneration of adrenal cortex (f);

clinical signs.
100, 200 and 400 mg/kg bw/day, male and females: miosis
200, 400 mg/kg bw/day, male and female:: salivation
400 mg/kg bw/day, male and female: dirty fur
400 mg/kg bw/day, females: tremor before death
recovery period: 200, 400 mg/kg bw: clinical signs diappeared

BODY WEIGHT AND WEIGHT GAIN
400 mg/kg bw/day males: significantly depressed body weight.
274 g versus 334 g in control, no further information
recovery period: 361 g versus407 g in control , no further information

CLINICAL CHEMISTRY
200 mg/kg bw/day, male: not significant increase in total cholesterol which normalized during recovery period
72 mg/dl versus 51 mg/dl in controls
recovery period 53 mg/dl versus 57 mg/dl in controls
200 mg/kg bw/day, males, recovery period:
decreased Albumin/ Globulin ratio
1.06 (p<=0.05) versus 1.18 in controls
female, 200 mg/kg bw/day: significant increase in blood glucose which normalized during recovery period
116 mg/dl (p<=0.01) versus 91 mg/dl in controls
recovery period: 126 mg/dl versus 116 mg/dl in controls


ORGAN WEIGHTS (significant changes only)
males: 200 mg/kg bw/day
significantly increased absolute spleen weight but not in relative weight:
0,63 g versus 0.51 g in controls which normaluized during rcovery period
during recovery period significant increased adernal weights
absolute. 62 mg (p>=0.01)versus 51 mg
relative: 0.016 (p<=0.05) versus 0.013
female, 200 mg/kg bw/day
significantly increased liver weights which normalized during recovery:
absolute: 7.07 g (p<=0.05) versus 5.91g in controls
relative 3.251(p<= 0.05) versus 2.939

HISTOPATHOLOGY: NON-NEOPLASTIC
suvivors showed no histopathological changes
Dose descriptor:
NOEL
Effect level:
50 other: mg/kg
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Critical effects observed:
not specified
RS-Freetext:
Mortality:
at 400 mg/kg bw 4/5 males and 5/5 females died within the treatment  period. Cause of deaths could not be determined.

Clinical signs:
100 mg/kg: m+f: miosis
200 mg/kg: m+f: miosis, salivation; reversible during                        recovery
400 mg/kg: m+f: miosis, salivation, dirty hair 
             f: premortal tremors
             m: reduced weight gain: ~20% after 4 weeks;                  partial recovery after end of treatment

Food consumption:
no effects

Hematology:
400 mg/kg: reduced white cell number (the 1 surviving male)

Clinical chemistry:
200 mg/kg: increased total cholesterol

Urinalysis:
100 mg/kg: increased urine volume (m)
200 mg/kg: increased urine volume (m+f)
400 mg/kg: increased urine volume (m)

Organ weights:
200 mg/kg: increased absolute weight (spleen, m; liver, f)
           increased relative weight (liver, f)
     after recovery:
           increased absolute weight (adrenals, m, slight)
           increased relative weight (adrenals, m, slight)
400 mg/kg: decreased absolute weight (spleen, m)

Histopathology:
Survivors: no effects
Deceased animals: inclusion bodies in hepatocytes (m+f); degeneration of  adrenal cortex (f);
Executive summary:

N,N-Dimethylbezylamine was given by gavage to male and female Crj:CD (SD) rats in doses of 0 (corn oil), 50, 100, 200, 400 mg/kg bw/day diluted in corn oil over a period of 28 days. In addition, animals of the control group and animals treated with 200 and 400 mg/kg bw/day were obseserved after termination of treatment for further 2 weeks (recovery group). Mortalities of both males and females receiving 400 mg/kg were observed from week 2. Clinical observation revealed miosis in both sexes receiving 100 mg/kg and miosis and salivation in those receiving 200 and 400 mg/kg. Body weight gain was suppressed in males receiving 400 mg/kg. Slight increases in total cholesterol were observed in males receiving 200 mg/kg. Pathological examination revealed no abnormalities attributable to the test substance treatment. The NOEL for repeat dose toxicity is considered to be 50 mg/kg/day for both sexes (MHLW 1997).

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 July 2022 to March 2023
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
June 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Strain: Wistar Han Rats
Substrain: Crl:WI(Han)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 6-7 weeks
- Weight at study initiation: males: 117 – 189 g; females: 101 – 134 g
- Fasting period before study: none
- Housing: up to 5 animals of the same sex and same dosing group together
- Diet: SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany); ad libitum
- Water: ad libitum
- Acclimation period: 13 days

DETAILS OF FOOD AND WATER QUALITY:
- no known contaminants in the feed that would interfere with the objectives of the study; phytoestrogen content: 290.69 mg TGE/kg (food batch used during the in-life phase); 440.91 mg TGE/kg (food batch used during acclimatization period)
- Municipal tap water.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): daily mean temperature during the study period: 20 to 22°C
- Humidity (%): daily mean relative humidity: 48 to 61%
- Air changes (per hr): at least 10 per hour
- Photoperiod (hrs dark / hrs light): 12-hours light and 12-hours dark

IN-LIFE DATES: From: 26 July 2023 To: 09 November 2023
Route of administration:
oral: gavage
Vehicle:
other: 1% aqueous carboxymethyl cellulose + 0.1% Tween80
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: at least weekly

VEHICLE
- Justification for use and choice of vehicle (if other than water): addition of CMC and Tween80 for a stable suspension
- Concentration in vehicle: 0, 15, 30, 50 mg/mL
- Amount of vehicle (if gavage): 5 ml/kg
- pH of formulation was adjusted to pH 9±0.1 with 37% HCl (only for dose formulation, not for vehicle formulation)
Analytical verification of doses or concentrations:
yes
Remarks:
Analysis was performed with an ultra performance liquid chromatographic method with tandem mass spectrometry (UPLC-MSMS) during weeks 1 to 6 and with UPLC with spectrophotometric detection (UPLC-UV) from week 9 onwards.
Details on analytical verification of doses or concentrations:
The concentrations analyzed in the formulations of Week 1 (Groups 2 and 4), Week 9 (Groups 2, 3 and 4) and Week 12 (Groups 2, and 3) were in agreement with target concentrations (i.e., mean sample concentration results were within or equal to 85-115% of target concentration).
For the formulation of Group 3 prepared for use in Week 1, the mean concentration was 74%. Results from re-diluting and reanalysis in Weeks 2 and 5 were not accepted due to issues with the analytical method. For the formulation of Groups 2, 3 and 4 prepared for use in Week 6, analysis was performed, but the results were also not accepted due to issues with the analytical method.
In the Group 1 formulations prepared for use in Weeks 1 and in Week 6, no test material was detected. Small responses at the retention time of the test material were observed in the chromatograms of the Group 1 formulation prepared for use in Week 9 and in Week 12. The maximum contribution to the Group 2 sample was 0.33%. It was considered not to derive from the formulation since a similar response was obtained in the analytical blanks.

The formulations of Group 2 (Week 1, 9 and 12), Group 4 (Weeks 1 and 9) and Group 3 (Weeks 9 and 12) were homogeneous (i.e. coefficient of variation ≤ 10%).
Duration of treatment / exposure:
90 days
Animals of Group 4 were sacrificed prematurely on Day 52 based on premature deaths and severe clinical signs.
Frequency of treatment:
daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
75 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on results of a 14-day repeated dose toxicity study with oral exposure of N,N-Dimethylbenzylamine in rats.
- Fasting period before blood sampling for clinical biochemistry: yes (overnight with a maximum of 24 hours)
- Rationale for selecting satellite groups: no satellite group
- Post-exposure recovery period in satellite groups: no recovery group
- Dose range finding studies: 14 days repeated dose dose range finding study (Test Facility Study No. 20342598)
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily; from Day 1 at 45 min to 75 min postdose.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly; from Week 1 and throughout the study, and on the day of necropsy

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined: Yes

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

WATER CONSUMPTION AND COMPOUND INTAKE: No
- Water consumption was monitored by visual inspection of the water bottles.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: once during pretreatment period and once during Week 13
- Dose groups that were examined: all animals during pretreatment period; initially only Group 1 and 3 animals during Week 13; due to findings in Group 3 aninmals, group 2 animals were also examined

HAEMATOLOGY: Yes
- Time schedule for collection of blood: between 7.00 and 10.30 at the end of the study
- Anaesthetic used for blood collection: Yes (isoflurane )
- Animals fasted: Yes (overnight)
- How many animals: all

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: between 7.00 and 10.30 at the end of the study
- Animals fasted: Yes (overnight)
- How many animals: all

SERUM HORMONES: Yes (T3, T4, TSH)
- Time of blood sample collection: between 7.00 and 10.30 at the end of the study
- Animals fasted: Yes (overnight)
- How many animals: all

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once during the dosing period
- Dose groups that were examined: the first 5 animals per sex per group
during Week 12-13
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: No

OTHER:
Estrous Stage Determination
- Time schedule for examinations: end of treatment - on the day of necropsy
- Dose groups that were examined: all
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Necropsy with evaluation of the carcass and musculoskeletal system including all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues.

Organ weights recording.

HISTOPATHOLOGY: Yes according to guideline requirements

Optional endpoint(s):
Optional endpoints: Yes
- sperm number
- sperm motility and progressive motility
- sperm morphological evaluation
- How many animals: all males with scheduled sacrifice
Statistics:
Means, standard deviations (or % coefficient of variation or standard error, when deemed appropriate), percentages, numbers, and/or incidences were reported as appropriate by dataset.
All statistical tests were conducted at the 5% significance level. All pairwise comparisons (individual dose groups versus control group) were conducted using two sided tests and were reported at the 1% and 5% levels, unless otherwise noted.

Parametric/non-parametric statistical methods were applied for the following variables: body weight, body weight gains, hematology variables, coagulation variables, clinical chemistry variables, FOB quantitative variables, organ weights, organ weight relative to body weight.

Parametric/Non-parametric analysis:
Levene’s test was used to assess the homogeneity of group variances.
The groups were compared using an overall one-way ANOVA F-test if Levene’s test was not significant or the Kruskal-Wallis test if it was significant. If the overall F-test or Kruskal-Wallis test was found to be significant, then pairwise comparisons were conducted using Dunnett’s or Dunn’s test, respectively.
For the motor activity data set parametric (ANOVA) tests on group means were with Bonferroni correction for multiple testing. Mixed modelling techniques, comparing six different covariance structures, were used in order to select the best fitting statistical model.

Incidence analysis:
A Fisher’s exact test will be used to conduct pairwise group comparisons of interest.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 75 mg/kg/day slight salivation was occasionally noted in three males and six females between Days 24 and 90. In addition, red discharge from the mouth was noted incidentally in one male and five females between Days 42 and 92.
At 150 mg/kg/day, generalized slight tremors were observed in two males and four females. For most animals the concurrency was low, observed only up to three times, but for male No. 21, the tremors were observed up to fifteen times. Tremors were observed between 45-75 minutes post dose. In addition, slight to severe salivation from Day 18 onwards, and red discharge from the mouth from Day 32 onwards was observed in all males and females. These clinical signs were observed on a regular basis, but not on all days.
Erected fur in three males between Days 53 and 83, decreased activity in one male on Days 54 55, hunched posture in one female on Day 61 and abnormal breathing sounds in one female on Day 45 were also noted.
Any other clinical signs noted during the treatment period 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 and did not show any apparent dose-related trend. At the incidence observed, these were considered not to be test material-related.

The animal of Group 3 (150 mg/kg bw/d) found dead was observed with salivation and red discharge from the mouth between Days 30 and 40. No effects on body weight or body gain were noted.
The following clinical signs were observed in the five animals of Group 4 (250 mg/kg bw/d) found dead: generalized tremors (up to severe) from Day 3 onwards, salivation (up to moderate) from Day 9 onwards and red or yellow discharge from the mouth and/or nostrils from Day 28 onwards. In addition, abnormal gait, decreased activity, abnormal breathing sounds, labored and/or shallow breathing, erected fur and/or hunched posture were observed immediately and/or 45-75 minutes post dosing.
Clinical signs in Group 4 (250 mg/kg bw/d) animals sacrificed prematurelly comprised generalized tremors (up to severe), salivation (up to severe), red/yellow discharge from the mouth, hunched posture, erected fur, labored breathing or abnormal breathing sounds, decreased activity, abnormal gait and/or weak appearance.
Mortality:
mortality observed, treatment-related
Description (incidence):
At 150 mg/kg/day, Male No. 30 was found dead on Day 42. There were no gross observations, and no cause of death could be determined from the sections examined at histopathology. A test material-related cause of death could not be excluded.

At 250 mg/kg/day, five animals were found dead between Days 31 and 50 (Male Nos. 39 and 40 and Female Nos. 73, 76 and 78 on Days 44, 31, 45, 47 and 50, respectively). The cause of death was undetermined based on the histopathological sections examined, but was regarded to be test material-related.
All remaining animals at 250 mg/kg/day were sacrificed on Day 52, based on the mortality in this dose group and the observed severe clinical signs.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weights and body weight gain of animals dosed at 75 and 150 mg/kg/day were considered to have been unaffected by treatment with the test material. The higher body weight in males at 75 mg/kg/day (8% of control) was considered to be not test material-related in absence of a dose-related response. At 150 mg/kg/day, the body weight was mostly below the body weight of the control group, but not more than -5 and -7% for males and females, respectively.
Any statistically significant changes in body weight gain were considered to be unrelated to the test material since no trend was apparent regarding dose and duration of dosing.
Animals of group 4 found dead or sacrificed prematurely did not reveal any effects on body weight or body weight gain.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption of males and females at 75 mg/kg/day was considered to have been unaffected by treatment with the test material.

Lower food consumption was noted in males at 150 mg/kg/day between Days 1-36 (decreased by 10%) and 64 71 (decreased by 14%), while overall food consumption was comparable to control (4% lower than control). For females, lower food consumption was noted at 150 mg/kg/day between Days 1 57 (up to 15% lower than control) and 71-91 (decreased by 6%), which resulted in a lower overall food consumption (9% lower than control). As the lower food consumption observed in males and females at 150 mg/kg/day was without an effect on body weight and body weight. Due to the low severity and without effects on body weight, this was considered to be non-adverse.

No clear effect on food consumption was observe in animals of Group 4 (250 mg/kg bw/day) sacrificed on Day 52.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
effects observed, treatment-related
Description (incidence and severity):
Corneal edema (up to moderate) was observed in one or both eyes in one control male, but not in control females, four males and five females at 75 and 150 mg/kg/day each during the ophthalmic examinations in Week 13. For one male and one female at 150 mg/kg/day this finding was also observed during the pretreatment examination. In one male at 150 mg/kg/day, the edema in one eye was scored as severe, and moderate vitreous hemorrhage was also noted.
The ophthalmic corneal edema, observed in males and females at 75 and 150 mg/kg/day, and vitreous hemorrhage observed in one male at 150 mg/kg/day, occurred without a microscopic correlate, and was therefore considered to be non-adverse.

The nature and incidence of the other ophthalmology findings noted during the pretreatment period and in Week 13 was similar among the groups, and occurred within the range considered normal for rats of this age and strain. These findings were therefore considered to be unrelated to the test material.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No test material-related changes were noted in hematological parameters in males up to 150 mg/kg/day.
In females, a lower mean corpuscular hemoglobin concentration was noted at 150 mg/kg/day (0.98x of control), which was just outside the historical control data. As the control group was on the low end of the historical control data, and in absence of a histopathological correlate, this finding is considerd non-adverse. Additionally, coagulation parameters were considered not to have been affected by treatment with the test material up to 150 mg/kg/day. No clear effect on hematology parameters were observed in Group 4 animals at premature sacrifice.

Remaining differences in hematology parameters, regardless of statistical significance, were considered not test material-related based on the absence of a dose response, general overlap of individual values with the range of control values, and/or were of a magnitude of change commonly observed in rats under similar study conditions.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
In females, a lower concentration was observed in total bilirubin at 150 mg/kg/day (0.80x of control) and in creatinine at 75 and 150 mg/kg/day (0.89x for both). In addition, in females a higher concentration of potassium at 75 and 150 mg/kg/day (1.04 and 1.10x, respectively) and chloride at 75 and 150 mg/kg/day (1.02x for both) was observed. All values remained within the range of historical control values .
Remaining differences in clinical chemistry parameters, regardless of statistical significance, were considered not test material-related based on the absence of a dose response, general overlap of individual values with the range of control values, and/or were of a magnitude of change commonly observed in rats under similar study conditions.
Endocrine findings:
no effects observed
Description (incidence and severity):
No effects on hormone analysis were observed in females dosed with the test material.
In males, T3 levels were higher at 75 and 150 mg/kg/day (1.32 and 1.55x of control, respectively, not statistically significant at 75 mg/kg/day) and T4 levels were higher at 150 mg/kg/day (1.21x, not statistically significant). All mean values remained within the historical control range . TSH values were unaffected. No clear effects were observed in prematureley sacrificed animals of Group 4.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Functional observations (activity, excitability, physiological and sensorimotor) were considered to be unaffected by treatment with the test material up to 150 mg/kg/day.
On autonomic functional observations, erected fur was present in 5/5 males at 75 and 150 mg/kg/day compared to 2/5 control animals, and severe salivation was present in 1/5 females at 75 mg/kg/day compared to 0/5 controls.
On neuromuscular functional observations, a slightly impaired gait/mobility was observed in 1/5 females at both 75 and 150 mg/kg/day compared to 0/5 control animals.
Remaining findings in functional observations were considered not test material-related based on the absence of a dose response, and general overlap of individual values with the range of control values.
Motor activity was similar between treated and control groups. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.
Functional observations were not performed on the found dead and prematurely euthanized animals at 250 mg/kg/day.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Higher liver weights were recorded for males and females at 150 mg/kg/day (relative to body weight).
Lower thymus weights were recorded for females at 150 mg/kg/day (absolute and relative to body weight).
There were no other test material-related organ weight changes.
Remaining organs weight changes which reached statistical significance were noted in males only and included higher pituitary gland weight at 150 mg/kg/day (relative to body weight only), lower prostate gland weight at 75 mg/kg/day (relative to body weight only) and higher testes weight at 75 mg/kg/day (absolute only). These were regarded unrelated to the treatment with the test material as they lacked a dose-related pattern and/or remained within the range considered normal for rats of this age and strain.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test material-related gross observations at the end of the 90-day treatment period up to 150 mg/kg/day.
All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Macroscopically, two animals of Group 4 found dead were observed with a thick or enlarged liver (Male No. 40 and Female No. 76, respectively). During macroscopic examination of the animals sacrificed prematurely, an irregular surface was observed in the non-glandular stomach of 6/8 males and 3/7 females at 250 mg/kg/day.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In the liver, centrilobular hepatocellular hypertrophy was recorded at minimal degree in 2/9 males and 5/10 females at 150 mg/kg/day. Effects observed in the liver were not accompanied by any degenerative or inflammatory lesions and were without alterations in liver enzymes and therefore regarded as non-adverse.
In the salivary glands, hypertrophy of the mucinous epithelium was recorded at 75 mg/kg/day at minimal degree in the mandibular gland in 1/9 males and 1/10 females and at 150 mg/kg/day in the mandibular gland (minimal-mild) in 9/9 males and 7/10 females and in the sublingual gland at minimal degree in 3/9 males and 2/10 females. These findings were, in absence of any degenerative or inflammatory lesions, regarded to be non-adverse.
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test material related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
In animals of Group 4 sacrificed prematurely hyperplasia of the squamous epithelium of the non-glandular stomach was observed in 2/8 males (minimal) and 2/7 females (mild). All animals of group 4 found dead showed centrilobular hepatocellular hypertrophy in the liver and one male revealed hypertrophy of the mucinous epithelium in the mandibular salivary glands. For the remaining animals of this group there was no microscopic correlate.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
The lower sperm count in the epididymis at 75 mg/kg/day was considered not test material-related based on the absence of a dose response, general overlap of individual values with the range of control values, and/ or were of a magnitude of change commonly observed in rats under similar study conditions .
A higher number of cells with normal morphology at 75 and 150 mg/kg/day, lower number of cells with coiled tails at 75 and 150 mg/kg/day, and lower number of cells with abnormal head at 75 mg/kg/day were observed compared to concurrent controls. These observations were considered not test material related as an opposite effect would be expected in case of toxicity.
Details on results:
see attached summary tables for
- clinical observations
- body weights
- body weight gains
- food consumption
- functional observations
- motor activity test
- hematology values
- coagulation values
- clinical chemistry values
- sperm analysis
- organ weights
- macroscopic pathology
- microscopic pathology
Dose descriptor:
NOAEL
Effect level:
75 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
other: mortality
Dose descriptor:
LOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
other: mortality
Critical effects observed:
no
Conclusions:
At 250 mg/kg/day there was test material-related mortality, which was regarded adverse. In the remaining animals, adverse clinical signs of generalized tremors were observed. At 150 mg/kg/day, there was one spontaneous dead male of which a test material-related cause of death could not be excluded. In addition, adverse clinical signs of generalized tremors were observed in males and females.
From the results presented in this report a No Observed Adverse Effect Level (NOAEL) for N,N-Dimethylbenzylamine of 75 mg/kg/day was established.
Executive summary:

A 90-day repeated dose toxicity test was performed with Wistar rats which received 0, 75, 150 or 250 mg/kg bw/d of N,N-dimethylbenzylamine in 1% carboxymethyl cellulose with 0.1% Tween80 by gavage.


The following parameters and end points were evaluated in this study: mortality, clinical signs, body weight, food consumption, ophthalmology, functional observation tests, stage of estrous, sperm analysis, clinical pathology parameters (hematology, coagulation, clinical chemistry and hormone analysis), macroscopic examination, organ weights and microscopic examination.


At 75 mg/kg/day, occasional clinical signs of slight salivation and red discharge from the mouth in males and females were observed. Corneal edema was observed during ophthalmic examinations, and on functional observations erected fur and salivation (autonomic) and impaired gait/mobility (neuromuscular) were noted in males and/or females. These findings were considered to be non-adverse.
On clinical pathology, non-adverse lower creatinine and higher potassium and chloride concentrations in females and a higher T3 level in males were noted. On histopathology, non-adverse hypertrophy of the mucinous epithelium in a single male and female in the mandibular gland was noted.


At 150 mg/kg/day, one male was found dead on Day 42. Clinical signs observed prior to death were salivation and red discharge from the mouth. No other effects or gross observations were noted, and a test material-related cause of death could not be excluded.
On the remaining animals dosed at 150 mg/kg/day, adverse clinical signs of generalized tremors were observed in some males and females. In addition, non-adverse clinical signs of salivation and red discharge from the mouth in males and females, and incidentally erected fur and decreased activity in males, and hunched posture, and abnormal breathing sounds in females, were observed. A non-adverse lower food consumption was noted in males and females without an effect on body weight.
Corneal edema in males and females, and vitreous hemorrhage in a single male was observed during ophthalmic examinations, and on functional observations erected fur (autonomic) in males and impaired gait/mobility (neuromuscular) in a single female was observed. On clinical pathology, lower mean corpuscular hemoglobin concentrations, total bilirubin and creatinine concentrations and higher potassium and chloride concentrations in females were noted. These findings were considered to be non-adverse.
On histopathology, higher weights were recorded for the liver of both sexes (microscopic correlate: centrilobular hepatocellular hypertrophy) and lower weights were recorded for the thymus of females at 150 mg/kg/day (no microscopic correlate). Microscopic findings were present in the liver and consisted of centrilobular hepatocellular hypertrophy in two males and five females and in the salivary glands consisting of hypertrophy of the mucinous epithelium in all males and most females in the mandibular gland and in three males and two females in the sublingual gland. These test material-related morphologic alterations in the liver and salivary glands were regarded to be non-adverse.


At 250 mg/kg/day, five test material-related deaths were recorded, which was considered to be adverse. Clinical signs in these animals comprised generalized tremors, salivation, red or yellow discharge from the mouth and/or nostrils, abnormal gait, decreased activity, abnormal breathing sounds, labored/shallow breathing, erected fur and/or hunched posture. At macroscopy, two animals were observed with a thick or enlarged liver. Microscopic findings included centrilobular hepatocellular hypertrophy in the liver of all animals found dead and hypertrophy of the mucinous epithelium in the mandibular salivary glands in males.
Remaining animals at 250 mg/kg/day were sacrificed on Day 52, based on severe clinical signs of generalized tremors. In addition, clinical signs of salivation, red/yellow discharge from the mouth, hunched posture, erected fur, labored breathing or abnormal breathing sounds, decreased activity, abnormal gait and/or weak were observed.
On histopathological assessment, higher organ weights were observed in the adrenal gland and liver of males and females, and ovary/oviduct of females, and lower thymus weight was noted in females.
On macroscopic examination, an irregular surface was observed in the non-glandular stomach of males and females. The microscopic correlate for this finding was hyperplasia of the squamous epithelium of the non-glandular stomach in some males and females.


No test material-related changes were noted in any of the remaining parameters investigated in this study (i.e., body weight, functional observations (activity, excitability, physiological and sensorimotor), coagulation and sperm analysis).


In conclusion, Wistar Han rats were administered with N,N-Dimethylbenzylamine by once daily oral gavage for at least 90 days at 75 and 150 and mg/kg/day and for 52 days at 250 mg/kg/day. At 250 mg/kg/day there was test material-related mortality, which was regarded adverse. In the remaining animals, adverse clinical signs of generalized tremors were observed. At 150 mg/kg/day, there was one spontaneous dead male of which a test material-related cause of death could not be excluded. In addition, adverse clinical signs of generalized tremors were observed in males and females.


From the results presented in this report a No Observed Adverse Effect Level (NOAEL) for N,N-Dimethylbenzylamine of 75 mg/kg/day was established.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: guideline test and GLP
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
GLP compliance:
yes
Species:
rat
Strain:
other: Crl:CD(SD)BR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Weight at study initiation: males 158.5-184.5 g femalew:116.1-153.9 g
- Housing: 5
- Diet ad libitum
- Water ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 40-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
The test article was administered daily by gavage for 28 days
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
the achieved concentration was measured during week 1 and week 2 the analytical procedure HUK624/1-01F
Duration of treatment / exposure:
28 days
Frequency of treatment:
once daily
Remarks:
Doses / Concentrations:
0, 6, 30, 150 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
according to the respective OECD guideline
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily , full examination weekly

BODY WEIGHT: Yes
- Time schedule for examinations: once weekly

FOOD CONSUMPTION weekly:

WATER CONSUMPTION by visual appraisal:

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: week 4
- Anaesthetic used for blood collection: Yes (identity) / No / No data
- Animals fasted: Yes
- Parameters
hemoglobin conc., MCV,RBC, MCH, MCHC, PCV, WBC, platlet count
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: week 4
- Animals fasted: Yes
- Parameters
alkaline phosphatase, GOT/AST, GPT/ALT, blood urea, BUN, Cholesterol, creatininem glucose, total bilirubin, triglyceride,
albumin, albumin/globulin ratio,
total protein, Ca, K, Na, inorganic phosphorus

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Organ weights
adrenals liver kidneys testes
left and right organs were weighed separately

HISTOPATHOLOGY: Yes
tissues from control and high dose animals initially

tissue preservation
adremals, aorta, brain (medullary and cerebellar and cortical sections),
colon (duodenum, caecum, jejunum rectum), kidneys, liver, lungs, lymph nodes (mandibular and nesenteric) ,
eyes and optic nerve, oesophageousm pancreasm prostatem salivary glands, seminal vesicles, spinal cord, stomach,
thymus, trachea, uterus including cervix, ovaries, pituitary gland, femur with bone marrow, mammary gland, sciatic nerve,
skin, spleen, testes with epididymides, thyroid with parathyroids urinary bladder
Other examinations:
no data
Statistics:
anlysis of variance, t-test, Kruskal-Wallis test, Wilcoxon test
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
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):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
there were no clinical observations that were considered to be related to treatment
During week 4 1 control male died and in the intermediate dose 1 female
Both death were considered to result vrom blood sampling/anesthetic

BODY WEIGHT AND WEIGHT GAIN
The body weight gain of male and female rats was considered not to have been affected by treatment

HAEMATOLOGY and CLINICAL CHEMISTRY
The measured haematological and clinical chemistry parameters were considered to have remained unaffected by treatment

ORGAN WEIGHTS
Organ weights in test animals were in general comparable to organ weights of control animals

the absolute and relative testis weights for high dose group males were slightly higher han those of the controls.
However there was no histopathological correlate
Testis weights for the low and intermediate dose group males were similiar to those of the controls

GROSS PATHOLOGY and HISTOPATHOLOGY: NON-NEOPLASTIC
there were no histopathological findings of any unusual nature or incidence suggestive of target organ toxicity

Dose descriptor:
NOAEL
Effect level:
ca. 150 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: There were no major differences between control and treated rats. The high dose group males had slightly higher testis weights than the controls. However there was no histopathological correlate
Critical effects observed:
not specified

The data were processed, where appropriate, to give group mean values and standard deviations. The nature of the data was considered to be such that statistical analysis would not have provided additional information.

Executive summary:

In an test according to OECD TG 407 and GLP groups of male and female Crl:CD(SD)BR rats received N,N-Dimethylbenzylamine in corn oil by daily oral gavage at nominal dose levels of 6, 30 or 150 mg/kg bw/day. A further group of the same size received corn oil alone and served as control. There were no major differences between control and treated rats. The high dose group males had slightly higher testis weights than the controls. However there was no histopathological correlate. Therefore this finding was considered not to be of toxicological relvance. The NOAEL was determined to be 150 mg/kg bw/day (BG Chemie 1988).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
75 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The key studies are GLP-compliant and of high quality.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

ORAL ADMINISTRATION


Sub-acute studies:


1/ In a sub-acute oral GLP study performed according to the OECD TG 407, the test substance diluted in corn oil was administered by gavage to male and female Crl:CD(SD)BR rats at dose levels of 0, 6, 30, 150, 200 mg/kg bw/day (BG Chemie 1988, 1990).


There were no major differences between control and treated rats. The high dose group males had slightly higher testis weights than the controls. However there was no histopathological correlate. Therefore this finding was considered not to be of toxicological relevance. The NOAEL was determined to be 150 mg/kg bw/day.


2/ In another sub-acute oral GLP study performed according to Japanese guidelines, the test substance diluted in corn oil was administered by gavage to male and female Crj:CD(SD) rats at dose levels of 0, 50, 100, 200 and 400 mg/kg bw/day(MHLW, 1997).


Mortalities of both males and females receiving 400 mg/kg bw/day were observed from week 2. Clinical observation revealed miosis in both sexes receiving 100 mg/kg bw/day and miosis and salivation in those receiving 200 and 400 mg/kg bw/day. Body weight gain was suppressed in males receiving 400 mg/kg bw/day. Slight increases in total cholesterol were observed in males receiving 200 mg/kg bw/day. Pathological examination revealed no abnormalities attributable to the test substance treatment. The NOEL for repeat dose toxicity is considered to be 50 mg/kg bw/day for both sexes.


Sub-chronic study:


In a 90-day repeated dose toxicity test performed according to OECD TG 408 and in compliance with GLP, Wistar rats received 0, 75, 150 or 250 mg/kg bw/d of N,N-dimethylbenzylamine in 1% carboxymethyl cellulose with 0.1% Tween80 by gavage.


At 250 mg/kg/day there was test material-related mortality, which was regarded adverse. In the remaining animals, adverse clinical signs of generalized tremors were observed. At 150 mg/kg/day, there was one spontaneous dead male of which a test material-related cause of death could not be excluded. In addition, adverse clinical signs of generalized tremors were observed in males and females.


From the results presented in this report a No Observed Adverse Effect Level (NOAEL) for N,N-Dimethylbenzylamine of 75 mg/kg/day was established. This value is used as the basis for DNEL derivation.


 


DERMAL APPLICATION


There are no data available


 


INHALATION EXPOSURE


There are no valid studies available

Justification for classification or non-classification

Based on the available data, no classification is required according to the EU Regulation 1272/2008 (CLP/GHS).