Acacia mearnsi, ext., reaction products with ammonium chloride and formaldehyde

Endpoint summary

Description of key information

Oral Route:
A combined repeated dose toxicity study with the reproduction/ developmental toxicity screening has been conducted according to OECD 422 test guideline using the oral exposure route on wistar rats. 
No classification for repeat-dose toxicity is warranted based on the absence of toxicologically relevant effects in this study, according to the criteria of Annex VI Directive 67/748/EEC or the 1272/2008 regulation. 
Under the conditions of this study, no adverse systemic effects were observed and the No Observed Effect Level (NOEL) in males and females was considered to be 1000 mg/kg/day.  
Inhalation Route:
Based on the available data and in accordance with section 1 of REACH Annex XI, the repeat dose toxicity study via the inhalation route does not need to be conducted if the study does not appear to be scientifically necessary.
Dermal Route:
Based on the available data and in accordance with section 1 of REACH Annex XI, the repeat dose toxicity study via the dermal route does not need to be conducted if the study does not appear to be scientifically necessary.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study was conducted according to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines, which do not affect the quality of the relevant results.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Remarks:

No relevant deviation from the standard guideline

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Housing and Feeding Conditions
- Full barrier in an air-conditioned room
- Temperature: 22 3°C
- Relative humidity: 55 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice (lot no. 0702)
- Free access to tap water, (drinking water, municipal residue control, microbiological controls at regular intervals)
- The animals were kept individually in IVC cages (except during the mating period when one female will be paired with one male), type III H, polysulphone cages on Altromin saw fibre bedding (lot no. 010812)
- Certificates of food, water and bedding are filed at BSL BIOSERVICE
- Adequate acclimatisation period (at least 5 days) under laboratory conditions

Number and Sex of the Animals
80 animals (40 males and 40 females) were included in the study (10 male and 10 female animals per group).

Preparation of the Animals
Prior to the start of the treatment period a detailed clinical observation outside the home cage was made. Before the first administration all animals used for the study were weighed and assigned to the experimental groups with achieving a most homogenous variation in body weight throughout the groups of males and females.

Route of administration:

oral: gavage

Details on oral exposure:

Preparation of the Animals
Prior to the start of the treatment period a detailed clinical observation outside the home cage was made. Before the first administration all animals used for the study were weighed and assigned to the experimental groups with achieving a most homogenous variation in body weight throughout the groups of males and females.

Experimental Groups and Doses
A dose range finding study was performed to choose the dose (BSL Study number 122037) for the main study. Based on dose range finding study in which no overt toxicological changes were observed at 1000 mg/ kg bw/day and in consultation with the sponsor the following doses (Table 1) were selected for the 3 dose groups (LD = low dose, MD = medium dose, HD = high dose) and 1 control group (C).

The highest dose level was chosen with the aim of inducing toxic effects, but no death or severe suffering. Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dosage related response and NOAEL.

The animals in the control group were handled in an identical manner to the test group subjects and received the vehicle using the same dose volume.

Administration of Doses
The test item and vehicle were administered at a single dose to the animals by oral gavage using a gavaging cannula. The application volume for all groups was 5 mL/kg body weight.
For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured on week- based.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Preparation of the Test Item and Control Formulations
The test item was weighed into a tared plastic vial on a suitable precision balance and the sterile water was added to give the appropriate final concentration of the test item. The formulation was placed on Vortex machine for short period to ensure proper homogenistation of the formulation.
The vehicle has been selected as suggested by the sponsor and on the basis of the test item’s characteristics.
The test item formulation was prepared freshly on each administration day before the administration procedure.

Dose Formulation Analysis
Each dosing concentration were analysed with respect to the target nominal concentration. Stability and homogeneity of the test item in the vehicle were analysed for the low and high dose concentrations.
Samples for the nominal concentration verification were taken in study week 1 (first week of pre mating period), 3 (first week of mating), 5 (gestation) and 7 (gestation/lactation).
Samples for homogeneity were taken from the top, middle and bottom of the high dose and the low dose preparation in study week 1 and 5.
Samples for stability analysis were taken in the first week of the study, 0 hours after the preparation and another sample 6 hours after the preparation (at room temperature), from high and low dose preparations.
All formulation samples were preserved at -20 oC until the analysis. The samples were analyzed at Spectral Service AG, Emil-Hoffmann-Str. 33, 50996 Köln, Germany. The contents of the reaction products of acacia mearnsi with ammonium chloride and formaldehyde in the solutions will be determined by 1H-NMR using caliberation curve of test item and internal standards. This phase of the study was performed as standalone GLP study and the procedure followed for the sample analysis was described in detail in the study plan issued by Spectral Service.

Duration of treatment / exposure:

The animals were treated with the test item formulation or vehicle on 7 days per week for a period of 54 days, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 3 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days (5 males from each group received dose applications for 28 days and the rest 5 animals from each group received dose applications for 29 days) was completed.

Frequency of treatment:

Once daily

Remarks:

Doses / Concentrations:
1000 mg/kg/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
300 mg/kg/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
100 mg/kg/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
0 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

10 animals per sex per dose.

Control animals:

yes, concurrent vehicle

Details on study design:

According to the results of the dose range finding study and in consultation with the sponsor three selected doses were tested for the 3 dose groups (LD = low dose, MD = medium dose, HD = high dose) and 1 control group (C).

Observations and examinations performed and frequency:

Clinical Observations
General clinical observations were made once a day in all animals. Pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity, including mortality were recorded.
Once before the first exposure, and once a week thereafter, detailed clinical observations were made in all animals outside the home cage in a standard arena. Clinical observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour were recorded.

Functional Observations
Multiple detailed behavioural observations were made in the week before the first treatment and during the last week of the treatment in 5 selected males and on day 3 of the lactation period in 5 selected females (only lactating females were evaluated) from each of control and treated groups outside the home cage using a functional observational battery of tests:
Sensory reactivity to different modalities, grip strength and motor activity assessments and other behavioural observations as well as rearing supported and not supported, urination, defecation, startle/ auditory response, equilibrium reflex, positional passivity, visual placing, fore and hind limb grip strength, tail pinch response, toe pinch reflex, extensor thrust/limb tone, hind limb reflex, righting reflex on the ground, air righting reflex, pupil response, body temperature and ophthalmoscopy (anterior chamber of the eye and fundus of eye).

Haematology
Haematological parameters from 5 selected males and females from each group (C, LD, MD and HD groups) were examined at the end of the treatment prior to or as part of the sacrifice of the animals.
Blood from the abdominal aorta of the animals was collected in EDTA-coated tubes.
The following haematological parameters were examined: haematocrit value , haemoglobin content, red blood cell count , mean corpuscular volume (, mean corpuscular haemoglobin , mean corpuscular haemoglobin concentration , reticulocytes , platelet count , white blood cells , neutrophils , lymphocytes, monocytes, eosinophils, basophils

Blood Coagulation
Coagulation parameters from 5 selected males and females from each group (C, LD, MD and HD groups) were examined at the end of the treatment prior to or as part of the sacrifice of the animals.
Blood from the abdominal aorta of the animals was collected in citrate-coated tubes (prothrombin time; activated partial thromboplastin time)

Clinical Biochemistry
Parameters of clinical biochemistry from 5 selected males and females from each group (C, LD, MD and HD groups) were examined at the end of the treatment prior to or as part of the sacrifice of the animals.
Blood from the abdominal aorta of the animals was collected in serum separator tubes.
The following parameters of clinical biochemistry were examined :
alanine aminotransferase (ALAT)
aspartate-aminotransferase (ASAT)
alkaline phosphatase (ALP)
creatinine (Crea)
total protein (TP)
albumin (Alb)
urea
total bile acids (TBA)
total cholesterol (Chol)
glucose (Gluc)
sodium (Na)
potassium (K)

Urinalysis:
A urinalysis was performed with samples collected from 5 selected males from each group (C, LD, MD and HD groups) prior to or as part of the sacrifice of the animals. Additionally, urine colour/ appearance were recorded.
The following parameters were measured using qualitative indicators :
specific gravity
nitrite
ph-value (pH)
protein
glucose
ketone bodies (ketones)
urobilinogen (ubg)
bilirubin
blood
leukocytes

Sacrifice and pathology:

Pathology
All animals were subjected to a detailed gross necropsy which includes careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.

Statistics:

The findings of this study were evaluated in terms of the observed effects, the necropsy and the microscopic findings.
The evaluation included the relationship between the dose of the test item and the presence or absence, incidence and severity of abnormalities, including gross lesions, identified target organs, infertility, clinical abnormalities, affected reproductive and litter performance, body weight changes, effects on mortality and any other toxic effects.
Parameters like body weight gain and food consumption were calculated for each animal as the difference in weight measured from one week to the next. Mean body weights and mean food consumptions were also presented as figures. The relative organ weights were calculated in relation to the body weight (measured at necropsy) and are presented as percentage.
Findings which were categorised into slight, moderate, marked and severe were not related to levels of statistical significance, but related to levels of biological relevance in consideration of the normal variation of the respective parameter (historical data) and also taking into account the respective values of the control animals of the study.
A statistical assessment of the results of the body weight, food consumption, parameters of haematology, blood coagulation and clinical biochemistry and absolute and relative organ weights were performed for each gender by comparing values of dosed with control animals of the main groups using a one-way ANOVA and a post-hoc Dunnett Test. These statistics were performed with GraphPad Prism V.5.01 and V.6.01 software (p<0.05 was considered as statistically significant).

Clinical signs:

no effects observed

Description (incidence and severity):

see "Details on results".

Mortality:

no mortality observed

Description (incidence):

see "Details on results".

Body weight and weight changes:

no effects observed

Description (incidence and severity):

see "Details on results".

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

see "Details on results".

Food efficiency:

not specified

Description (incidence and severity):

see "Details on results".

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

not specified

Description (incidence and severity):

see "Details on results".

Ophthalmological findings:

no effects observed

Description (incidence and severity):

see "Details on results".

Haematological findings:

no effects observed

Description (incidence and severity):

In the absence of dose response pattern the finding was not related to treatment.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

The statistical evaluation of data revealed no significant differences between the values of treated and control groups.

Urinalysis findings:

no effects observed

Description (incidence and severity):

see "Details on results".

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

see "Details on results".

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The above changes in organ weight (both males and females) data were not confirmed by histological changes. Hence, the findings were not considered to have toxicological relevance.

Gross pathological findings:

no effects observed

Description (incidence and severity):

see "Details on results".

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Other histopathological changes seen at terminal sacrifice were considered to be incidental in origin and/or within the range of expected changes for rats of this age and strain kept under laboratory conditions

Details on results:

Mortality
No mortality occurred in the control or treated groups during the treatment period.

Clinical Observations
There were clinical signs namely nasal discharge (Males C: 3/10, MD: 2/10; HD: 1/10; females MD: 2/10), alopecia (Males LD: 1/10; Females HD: 1/10), chromodacryorrhea (Males C: 1/10), injury (Males HD: 2/10; Females C: 1/10), moving the bedding (Males MD: 1/10), abnormal breathing (Males HD:1/10; Females LD: 1/10, MD: 1/10, HD: 2/10), salivation (Females MD: 1/10, HD: 3/10), piloerection (Females MD: 2/10, HD: 2/10), wasp waist (Females MD: 1/10), reduced spontaneous activity (Females MD: 1/10), trembling before application (females HD: 1/10) recorded in male or female animals of treated or control groups. These clinical signs were noted in few isolated animals occasionally and transiently. These findings were not considered to be of toxicological relevance.
During the weekly detailed clinical observation, no significant changes or differences between the groups were found.
There were no ophthalmoscopic findings in any of the animals of this study.

Functional Observations
No relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period. There were no biologically relevant differences in body temperature between the groups.

Body Weight Development
In both males and females, no treatment related changes were noted for body weight and body weight gain during the study period. Statistically there were no significant differences noted between the treated and corresponding controls. However, there was substantial decrease (lower by 138.29 % over control group) in body weight gain noted in females between the lactation days 0-4 in HD group. In the absence of statistical significance this finding was not considered to be of toxicological relevance.

Food Consumption
In both males and females, no treatment related changes were noted for treated group when compared to corresponding control. Statistically there were no significant differences noted between the treated and corresponding controls.

Haematology and Coagulation
There were no treatment related changes noted for haematological and coagulation parameters in male and female treated groups when compared to the corresponding control. However, there was statistical significant increase (higher by 18.64 % over control group) in mean aPTT value in male HD group, but this being within the historical control range was not considered to be due to treatment. There was statistically significant decrease (lower by 7.32 % over control group) in mean PT value of female MD group. In the absence of dose response pattern the finding was not related to treatment.

Clinical Biochemistry
There were no treatment related changes considered for measured clinical biochemistry parameters (ASAT, ALAT, ALP, CREA, TP, Alb, Urea, Chol, Glu, Na, K and TBA) of male and female treated groups when compared to corresponding control. The statistical evaluation of data revealed no significant differences between the values of treated and control groups.

Urinalysis
The urinalysis performed in male animals revealed no treatment related effect in treated groups when compared to the control group.

Pathology
At terminal sacrifice, macroscopic organ findings noted were few, and none of them was considered to be test item-related.
Yellow spot(s) were observed at the epididymis in a number of control and treated rats, but were not considered test item-related as histologically they were confirmed to represent spermatic granuloma, a finding occasionally seen in untreated rats of this strain and age.

Organ Weight
In males, there was statistically significant decrease noted for absolute (lower by 13%) and relative (lower by 12%) prostate weight in LD group. In addition there were slight decrease noted for absolute (lower by 17%) and relative (lower by 15%) spleen weight in HD group; slight to moderate decrease of absolute (lower by 26 and 14%) and relative (lower by 25 and 11%) thymus weight in LD and HD groups and moderate increase in absolute (higher by 25%) thymus weight in MD group; slight decrease in relative (lower by 20%) pituitary weight in MD group without attaining the statistical significance.
In females, there was statistically significant decrease noted for absolute (lower by 19%) left adrenal weight in MD and HD groups. In addition, there was slight decrease or increase in relative left (lower by 16% in MD) or right (higher by 14% in HD) adrenal weight in MD or HD groups; slight decrease in absolute (lower by 20%) and relative (lower by 19%-20%) right and total adrenal weight in HD group; slight decrease noted for absolute (lower by 16%-18%) and relative (lower by 16%-20%) heart weight in LD, MD and HD groups; slight decrease in absolute (lower by 15%) and relative (lower by 17%) weight of pituitary gland in LD group and absolute (lower by 14%-MD and 17% in LD ) and relative (lower by 17% in LD) thymus weight in LD or MD groups. There was slight increase in absolute (higher by 20%) uterus weight in LD group and moderate increase in absolute (higher by 28%) and relative (higher by 33%) thyroid weight in MD group without attaining the statistical significance.
The above changes in organ weight (both males and females) data were not confirmed by histological changes. Hence, the findings were not considered to have toxicological relevance.

Histopathology
No clear histopathological findings of toxicological significance were noted in the organs evaluated in randomized males and females of the control and high dose group.
In the liver, a minimal centrilobular hepatocellular hypertrophy was seen in 3/5 males of HD group. In the absence of concomitant organ weight changes, this was considered to be a chance event and to be unrelated to the treatment.
In the brain, a neoplastic lesion (oligodendroglioma) was observed in 1/5 male of HD group, and mild focal gliosis occurred in 1/5 female of the same dose group. Both lesions are rare, but may be occur spontaneously in rats of this strain and age. They were therefore considered to represent most probably chance events, although a relationship to the test item could not be completely excluded.
In the nonglandular part of the stomach (forestomach), the incidence and mean degree of severity of epithelial hyperplasia of the limiting ridge was mildly increased in males, but not in females of HD group. This inter-group difference was considered to be indicative of a minor local irritant effect of the test item formulation in the stomach, under the conditions of this study, and therefore to be without toxicological significance.
Other histopathological changes seen at terminal sacrifice were considered to be incidental in origin and/or within the range of expected changes for rats of this age and strain kept under laboratory conditions.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Critical effects observed:

not specified

Conclusions:

In conclusion, the repeated dose administration of the Acacia mearnsi, ext., reaction products with ammonium chloride and formaldehyde according to the 422 OECD guidance, to Wistar rats at different dosages revealed neither mortalities nor findings of toxicological relevance in animals.
Based on these results, the no observed adverse effect level (NOAEL) for systemic is considered to be 1000 mg/kg body weight/ day.
According to the 1272/2008/EC the substance shouldn't need to be classified.

Executive summary:

No mortality occurred in the control or treated groups during the treatment period of this study.

Slight clinical signs were observed during the treatment period in the control and treated groups (LD, MD and HD groups), which were not considered to have toxicological relevance.

During the weekly detailed clinical observation, no significant changes or differences between the groups were found. There were no ophthalmoscopic findings in any of the animals of this study.

No relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period. There were no biologically relevant differences in body temperature between the groups.

In both males and females, no treatment related changes were noted for body weight, body weight gain and food consumption during the study period.

There were no treatment related changes noted for haematological and coagulation parameters in treated groups when compared to the corresponding control.

There were no treatment related changes considered for measured clinical biochemistry parameters of treated groups when compared to corresponding control.

The urinalysis performed in male animals revealed no treatment related effect in treated groups when compared to the control group.

At terminal sacrifice, macroscopic organ findings noted were few, and none of them was considered to be test item-related. Yellow spot(s) were observed at the epididymis in a number of control and treated rats, but were not considered test item-related as histologically they were confirmed to represent spermatic granuloma, a finding occasionally seen in untreated rats of this strain and age.

Among the females one rat of MD group (No. 70) was found not to be pregnant. This was not dose-related and therefore considered to be unrelated to the treatment.

There were no changes considered to be of toxicological relevance noted for organ weight in both males and females when compared to corresponding control.

No clear histopathological findings of toxicological significance were noted in the other

organs evaluated in randomized males and females of the control and high dose group.

In the liver, a minimal centrilobular hepatocellular hypertrophy was seen in 3/5 males of HD group. In the absence of concomitant organ weight changes, this was considered to be a chance event and to be unrelated to the treatment.

In the nonglandular part of the stomach, epithelial hyperplasia of the limiting ridge was mildly increased in males of HD group, when compared to the controls. This was considered to be indicative of a minor local irritant effect of the test item formulation in the stomach and to be without toxicological significance.

In the brain, oligodendroglioma was observed in one male, and mild focal gliosis occurred in one female of HD group. These were considered to represent most probably chance events, although a relationship to the test item could not be completely excluded.

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:

Test was performed according to an international guideline (OECD 422) and according to GLP.

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

The animals were treated with the test item Acacia mearnsi extract formulation or vehicle on 7 days per week for a period of 54 days, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 3 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days (5 males from each group received dose applications for 28 days and the rest 5 animals from each group received dose applications for 29 days) was completed. Dose level: 100, 300 and 1000 mg/kg/day.

Based on the experimental conditions of this study, the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 1000 mg/kg/day in female and male rats based on systemic effect.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The only available study was performed according to the GLP and international guideline.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
A data waiver has been submitted to address this endpoint.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
A data waiver has been submitted to address this endpoint.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
A data waiver has been submitted to address this endpoint.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
A data waiver has been submitted to address this endpoint.

Justification for classification or non-classification

No classification for repeat-dose toxicity is warranted based on the absence of toxicologically relevant effects in this study -OECD 422 by oral route on Wistar rat according to the criteria of Annex VI Directive 67/748/EEC or Annex I regulation 1272/2008 regulation.

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