Registration Dossier

Administrative data

Description of key information

subchronic study in rat: NOAEL = 0.01% in diet (equivalent to 1.35 mg a.i./kg bw/d); distinct treatment related changes in ileum and mesenteric lymph nodes consisting of accumulations of large foamy macrophages at 0.1% and 0.3% in diet in both sexes.

chronic study in rat: NOAEL = 100 ppm in diet (1.03 mg a.i./kg bw/d for males and 1.38 mg a.i./kg bw/d for female); based on the alterations in the mesenteric lymph nodes observed for both sexes at 300 ppm in diet

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source and target substances have similar toxicological properties because
- they are manufactured from similar or identical precursors under similar conditions
- they share structural similarities with common functional groups (corresponding to scenario 2 of the read-across assessment framework): both, the target and source substance, are aliphatic amines with C8-18 alkyl chains and acetate functions
- Two thirds (w/w) of the target substance Reaction product of lauryl-PDA/lauryl-DETA with chloroacetic acid (excluding the solvent water) are composed of the source substance DOPA-Glycinate. The remaining third of Reaction product of lauryl-PDA/lauryl-DETA with chloroacetic acid consists of other aliphatic amines and derivatives which are considered as structural analogues to those constituting the source substance DOPA-Glycinate and may therefore be expected to elicit comparable (eco)toxicological effects.

The read-across hypothesis is based on structural similarity of target and source substances. Based on available experimental data, including key physicochemical properties and data from acute toxicity, repeated dose toxicity, genotoxicity and short term ecotoxicity studies, the read-across hypothesis is supported by a quite similar toxicological profile of both substances.

(Eco)toxicological, physicochemical and environmental fate data are summarised in the data matrix; robust study summaries are included in the Technical Dossier in the respective sections.

Therefore, read-across from the existing ecotoxicity, environmental fate and toxicity studies conducted with the source substances is considered as an appropriate adaptation to the standard information requirements of the REACH Regulation for the target substance, in accordance with the provisions of Annex XI, 1.5 of the REACH Regulation.

Further details are attached to IUCLID section 13.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
For further details refer to IUCLID section 13.

3. ANALOGUE APPROACH JUSTIFICATION
For further details refer to IUCLID section 13.

4. DATA MATRIX
For further details refer to IUCLID section 13.

Reason / purpose:
read-across: supporting information
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Species:
rat
Strain:
other: Sprague Dawley, Crl: CD(SD)BR
Sex:
male/female
Route of administration:
oral: gavage
Duration of treatment / exposure:
90 d
Dose descriptor:
NOAEL
Effect level:
12.5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
(20% aqueous solution, "product by process")
Sex:
male/female
Basis for effect level:
other: no adverse effects at highest administered dose
Key result
Dose descriptor:
NOAEL
Effect level:
2.5 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: no adverse effects at highest administered dose
Critical effects observed:
yes
Lowest effective dose / conc.:
20 mg/kg bw/day (actual dose received)
System:
immune system
Organ:
mesenteric lymph node
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
yes
Conclusions:
The oral 90 day NOAEL (rat, male/female) of the test item was 2.5 mg a.i./kg bw/d.
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
Study performance before implementation of the corresponding OECD guideline. However, study performance complies to some extent to the later implemented guideline.
GLP compliance:
no
Remarks:
Study performed before implementation of GLP
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
CIVO colony
Sex:
male/female
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
The test compound was thoroughly mixed into the diet by means of a mechanical blender (type Lödige, Paderborn). The diets were freshly prepared once a fortnight and stored at room temperature.

Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
continuously
Dose / conc.:
0.01 other: % in diet
Remarks:
corresponding to approx. 5 mg/kg bw/d (1.35 mg a.i./kg bw/d)
Dose / conc.:
0.03 other: % in diet
Remarks:
corresponding to approx. 15 mg/kg bw/d (4.05 mg a.i./kg bw/d)
Dose / conc.:
0.1 other: % in diet
Remarks:
corresponding to approx. 50 mg/kg bw/d (13.5 mg a.i./kg bw/d)
Dose / conc.:
0.3 other: % in diet
Remarks:
corresponding to approx. 150 mg/kg bw/d (40.5 mg a.i./kg bw/d)
No. of animals per sex per dose:
10
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: in a dose-range finding study the level of 1% resulted in mortali
ty,considerable growth depression, decreased food efficiency, increased water intake, increased haemoglobin content and red and white blood cell counts and 􀆧ncreased relative weight of kidneys. At 0.3% growth depression and decreased food efficiency was observed.
- Rationale for animal assignment (if not random): according to body weight
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Not specified

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: not specified

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption per group: during the first four weeks and during weeks 11 and 12

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: week 12
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Not specified
- How many animals: all animals
- Parameters: haemoglobin content, packed cell volume, red blood cell counts and total and differential white blood cell counts

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the study
- Animals fasted: Not specified
- How many animals: all animals
- Parameters: serum glutamic-pyruvic-transaminase (SGPT), serum glutamic-oxalacetic transaminase (SGOT), total serum protein, albumin and albumin-globulin ratio

URINALYSIS: Yes
- Time schedule for collection of urine: week 13
- Metabolism cages used for collection of urine: No; pooled samples were examined
- Animals fasted: Not specified
- Parameters: appearance, pH, glucose, albumin, occult blood, ketones and microscopy of the sediment

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No


Sacrifice and pathology:
GROSS PATHOLOGY: Yes


HISTOPATHOLOGY: Yes


Organ weights: heart, kidneys, liver, spleen, brain, testicles or ovaries, thymus, pituitary, thyroid and adrenal.

Detailed microscopic examination was carried out on all male and female rats of the highest dose group and of the control group.
Haematoxylin-eosin stained paraffin sections of the organs weighed and also of the following organs were examined: lung, trachea, spinal cord, salivary glands, exorbital lachrimal gland, prostate, epididymis, seminal vesicle, coagulating gland, uterus, urinary bladder, skeletal muscle, femoral nerve, thoracic aorta, esophagus, stomach, duodenum, ileum, caecum, colon, pancreas, skin, axillary and mesenteric lymph nodes, preputial gland and bone marrow (sternum) .
Microscopic examination of the rats fed on 0. 01, 0.03 and 0.1% of teh test item was restricted to spleen, kidneys, liver, mesenteric lymph nodes and gastro intestinal tract.
Frozen sections of a number of ilea and mesenteric lymph nodes were stained with Oil Red O. The alcian blue staining method for mucopolysaccharides, the PAS procedure and the Gram bacterial staining were applied to a number of selected ileum and mesenteric lymph node paraffin sections.
Clinical signs:
no effects observed
Description (incidence and severity):
No abnormalities of condition or behaviour were observed
Mortality:
no mortality observed
Description (incidence):
During the course of the experiment no death occurred.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Growth was significantly decreased in males of the highest feeding level. At 0.03 and 0.1% in males and at 0.3% in females average bodyweights were consistently lower than in controls, but the differences were
not statistically significant.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was similar apart from a slightly lower food intake at 0.3% in both sexes during week 11 and 12.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Food efficiency was decreased in males at 0.3%.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
White blood cell counts were considerably increased in both sexes at 0.3%. The differential counts showed an increase of neutrophilic cells with a decrease of lymphocytes in females at 0.3%. Abnormal looking
lymphocytes occurred in females at 0.03, 0.1 and 0.3%.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
SAP values tended to decrease with increasing feeding levels, but were only significantly decreased in females at 0.03, 0.1 and 0.3%. SGPT activities were distinctly decreased in females at 0.1 and 0.3% and
in males at 0.3%. Total serum protein and albumin content were decreased in males at the three highest feeding levels and in females at 0.3%. The albumin-globulin ratio was decreased at the highest feeding level in
both sexes. SGOT values did not show consistent differences between groups.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
In the urine sample of females at 0.3% some occult blood was noticed. Otherwise there were no alterations in the urine composition which could be attributed to the feeding of the test substance.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Kidney weights were significantly increased at 0.1 and 0. 3% in both sexes. Spleen weights were distictly increased at 0.3% in both sexes and slightly at 0.1% in males. The relative weight of the testicles was
increased at 0.3%.
No significant differences occurred in the other organs at any dose levels.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
At autopsy, the wall of the ileum was found to be slightly thickened and mesenteric lymph nodes appeared to be moderately enlarged at the two highest dose levels in both sexes. These findings were slightly more pronounced at 0.3% than at 0.1%.
Other autopsy findings, such as small greyish areas in the lung and proteinaceous plugs in the urinary bladder, are common findings in the strain of rats used. They may not be ascribed to the ingestion of the test material.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
At microscopic examination pathological changes related to the test item were observed in the ileum and mesenteric lymph nodes of males and females at 0.1% and 0.3%. In the ileum thick, club-shaped villi were observed. The surface epithelium was intact and the epithelial cells were normal in appearence. In the lamina propria large, foamy macrophages were seen. They had completely infiltrated the stroma, largely obscuring
the lymph and blood vessels. A small amount of separate, fine PAS- and alcian blue positive granules occurred within the foamy cytoplasm of these macrophages. Only a few macrophages were Oil-red-O positive. Ilea of some rats showed Gram-positive granules in the lamina propria. Small foci of polymorphnuclear leucocytes were seen in the villi of a few test animals, but other inflammatory cells were very rare as compared to controls.
The mesenteric lymph nodes revealed foamy macrophages scattered throughout the organ, containing fine PAS-, alcian blue and Gram-positive granules very similar to those observed in the ileum. Here, most macrophages were Oil-red-O negative. A necrotic area, surrounded by epitheloid-like cells, was observed in two males at 0.1%. Small accumulations of polymorphnuclear leucocytes were seen in a few males and females at 0.3%.
The microscopic changes described above showed a striking histological resemblance to Whipple's disease, a rare lesion in humans. These changes in ileum and mesenteric lymph nodes were slightly more pronounced at the 0.3% than at the 0.1% level in both sexes. They were completely absent at lower levels.
In the liver a colourless, crystalline substance, consisting of long, birefringent needles, was noticed in blood vessels and sinusoids. This phenomenon was not accompanied by a parenchymatous or a reticule-endothelial reaction of the liver. The crystalline substance was observed to a higher degree and frequency in the experimental animals than in controls, but was only pronounced in females at the 0.1% and 0.3% dose levels. The origin of this substance is not known. It is occasionally seen in rats of similar age as used in this study and the incidence seems to be attended with ageing. Therefore, no toxicological significance is attached to these changes.
Other microscopic changes occurred to about the same degree and frequency in test and control rats. Therefore they may not be ascribed to the ingestion of the test item.
Histopathological findings: neoplastic:
not examined
Dose descriptor:
NOAEL
Effect level:
5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
haematology
clinical biochemistry
Dose descriptor:
NOAEL
Effect level:
1.35 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
haematology
clinical biochemistry
Dose descriptor:
LOAEL
Effect level:
4.05 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
haematology
clinical biochemistry
Critical effects observed:
yes
Lowest effective dose / conc.:
13.5 mg/kg bw/day (nominal)
System:
other: gastrointestinal tract, lymph nodes
Organ:
ileum
mesenteric lymph node
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Results tables are attached as pdf below.

Conclusions:
The feeding of Reaction product of lauryl DPDA/DETA with chloroacetic acid (27% a.i.) to rats at various dietary levels up to 0.3% for three months resulted in distinct effects at 0.1 and 0.3%. The major abnormalities at 0.1 and 0.3% were deformed lymphocytes in the blood of females, decreased SGPT and SAP values, decreased total sorum protein and albumin content, increased relative weights of kidneys and spleen and pathological changes in ileum and mesenteric lymph nodes. At 0.03% the only difference with the controls consisted of the presence of deformed lymphocytes in females and decreased total serum protein and albumin content in males.
On the basis of the results, the no-toxic-effect level of Reaction product of lauryl DPDA/DETA with chloroacetic acid is placed at 0.01% in the diet of rats for three months, which is equivalent to 5 mg/kg bw/d in terms of test material or 1.35 mg a.i./kg bw/d.
Executive summary:

The toxicity of Reaction product of lauryl-PDA/lauryl-DETA with chloroacetic acid (27% aqueous solution) was examined in a sub-chronic (90-day) toxicity study with albino rats. The test material was fed at levels of 0, 0.01, 0.03, 0.1 and 0.3% in stock diet to groups of ten male and ten female weanling rats each. Observations were made of general appearance and behaviour, growth, food intake and efficiency, haematological factors and urine composition. Examinations were made on the blood serum for enzyme activities, viz, SGOT, SGPT, S.AP and for total serum protein and albumin.

At week 14 all rats were killed and examined grossly. Several organs were weighed and extensive histopathological examination was carried out.

 

At 0.3% growth depression, decreased food intake and food efficiency were observed in males. White blood cell counts were increased in both sexes at 0.3%. Differential counts showed a decrease of lymphocytes at 0.3% in females and abnormal looking lymphocytes in females at 0.03, 0.1 and 0.3%.

SGPT values were distinctly decreased at 0.3% in both sexes and at 0.1% in females. SAP activities were significantly decreased at the three highest feeding levels in females. Total serum protein and albumin content were decreased at 0.3% in both sexes and at 0.03 and 0.1% in males.

The relative weights of kidneys and spleen were increased at 0.3% in both sexes. Testicle weight was increased at 0.3%. At 0.1% kidney weight was increased in both sexes and spleen weight in males. At 0.03% all relative organ weights were within the normal range.

Gross and microscopic pathological examination revealed distinct treatment related changes in ileum and mesenteric lymph nodes consisting of accumulations of large foamy macrophages at 0.1% and 0.3% in both sexes.

It was concluded that the no-toxic-effect level of Reaction product of lauryl-PDA/lauryl-DETA with chloroacetic acid (27% aqueous solution) was 0.01% in the diet of rats for three months, which is equivalent to 5 mg/kg bw/d in terms of test material or 1.35 mg a.i./kg bw/d..

Endpoint:
chronic toxicity: oral
Remarks:
combined repeated dose and carcinogenicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
EU Method B.33 (Combined Chronic Toxicity / Carcinogenicity Test)
Deviations:
yes
Remarks:
see "Principles of method if other than guideline""
Principles of method if other than guideline:
The conduct of the study was consistent to EU method B.33 (88/303/EC) in all important aspects, with the following exceptions: 30 instead of 50 animals per sex and group were treated, no satellite groups were included, body weights were recorded biweekly instead of weekly during the first 12 weeks, food consumption was determined at biweekly intervals instead of weekly during the first 13 weeks, and full histopathology was not carried out on all animals of the control and the high dose group.
GLP compliance:
no
Remarks:
At the time of the study conduct, GLP was not compulsory.
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Central Institute for the Breeding of Laboratory Animals, TNO, Zeist, The Netherlands
- Age at study initiation: Not specified (newly weaned rats)
- Weight at study initiation: 81 g (male); 81–83 g (females)
- Fasting period before study: no data
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): no data

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
All diets were freshly prepared every 2–3 weeks and stored at ambient temperature.


Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
104 weeks
Frequency of treatment:
continuously
Remarks:
Doses / Concentrations:
30, 100 and 300 ppm in terms of test substance (27% aqueous solution)
Basis:
nominal in diet
No. of animals per sex per dose:
test group: 30 males and 30 females
control group: 50 males and 50 females
Control animals:
yes, plain diet
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: regularly

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: regularly

BODY WEIGHT: Yes
- Time schedule for examinations: biweekly during the first 12 weeks, at four-week intervals thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- at intervals during periods of two weeks

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 (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Number of animals: 10 rats per sex and group
- Time points: after 13, 26, 52, 78 and 102 weeks
- Parameters: haemoglobin content, packed cell volume, counts of erythrocytes and leucocytes, differential white blood cell count.

CLINICAL CHEMISTRY: Yes
- Number of animals: 10 rats per sex and group
- Time points: after 26, 52 and 104 weeks
- Parameters: blood glucose, blood urea nitrogen
- Number of animals: 5 rats per sex and group, 10 rats per sex and group
- Time points: after 26 and 52 weeks, at the end of the study (after 2 years)
- Parameters: serum glutamic-oxalacetic transaminase, serum glutamic-pyruvic transaminase, serum alkaline phosphatase, total serum protein and serum albumin

URINALYSIS: Yes
- Number of animals: 10 rats per sex and group (pooled samples)
- Time points: after 13, 26, 52, 78 and 103 weeks
- Parameters: appearance, pH, sugar, protein, occult blood, ketones and microscopic examination of the sediment
- Number of animals: 10 rats per sex and group
- Time points: after 13, 26, 52, 78 and 103 weeks
- Parameters: volume, specific gravity, glutamic-oxalacetic transaminase activity

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Organs: heart, kidneys, liver, spleen, brain, gonads, thyroid and adrenals

HISTOPATHOLOGY: Yes
- Macroscopic observations: all animals which died and all surviving animals.
- Histopathological examination (20 surviving males and 20 surviving females of the control and the highest treatment group): adrenals, aorta, brain, exorbital lachrymal glands, eyes, femoral nerve, gonads, heart, intestinal tract, liver, lung, lymph nodes (axillary and mesenteric), kidneys, mammary gland, oesophagus, pancreas, parathyroids, pituitary gland, preputial glands, prostate, salivary glands, skeletal muscle, skin, spinal cord, spleen, sternum with bone marrow, stomach, trachea, thymus, thyroid, urinary bladder, uterus and any other tissues which appeared abnormal on gross examination.
- Microscopic examination was performed on the mesenteric lymph nodes of all animals of all groups, except when this was prevented by advanced autolysis.
Statistics:
not specified
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:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
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:
no effects observed
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
The general health and the behaviour of treated and control rats were similar throughout the study.
No treatment-related effects on survival were observed during the study.

BODY WEIGHT AND WEIGHT GAIN
Mean body weights of male and female animals were comparable in all groups during the whole experimental period.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
No treatment-related effects on food consumption were observed during the study.

HAEMATOLOGY
After 102 weeks of exposure decreased values for haemoglobin content and packed cell volume were observed in high dose males and females. These findings were considered to be of minor toxicological significance, since similar effects did not occur during the first 1.5 years of the study and furthermore a shift towards lower haemoglobin levels and haematocrit values is commonly observed in older rats. No other effects on haematological parameters which may be attributed to treatment with the test item were observed.
Details are presented in Table A6.5- 1.

CLINICAL CHEMISTRY
No treatment-related changes were observed.
After 104 weeks of exposure, blood glucose levels were slightly but significantly decreased in females of all treatment groups. However, this finding is considered to be of no toxicological significance, since there was no evidence of a dose-related response and all values were within the normal range for the strain of rats used.
After 104 weeks of exposure, albumin levels were sightly but significantly decreased in high dose females. However, all values were within the normal range for the rat strain used.
Details are presented in Table A6.5- 1.

URINALYSIS
No treatment-related changes were observed.

ORGAN WEIGHTS
No treatment-related effects on organ weights were observed.

GROSS PATHOLOGY
No treatment-related gross lesions were recorded.

HISTOPATHOLOGY: NON-NEOPLASTIC
Histopathological examinations revealed treatment-related changes in the mesenteric lymph nodes of high dose animals only. These findings comprised aggregates of macrophages often showing signs of degeneration and necrosis, in a few rats accompanied by disturbance of lymph node architecture. The increased incidences of animals showing small numbers of macrophages in the mesenteric lymph nodes without evidence of cell or tissue damage observed in low and mid dose animals was considered to be an adaption to an increased demand rather than the expression of an untoward effect, since this alteration was also observed in the controls. Results are presented in Table A6.5- 1.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
No treatment-related neoplastic lesions were observed. The most frequently observed neoplasms were pituitary chromophobe adenomas, adrenal phaeochromocytomas, and tumours of the mammary glands. The tumours were approximately equally distributed amongst the controls and test animals, without any evidence of treatment-related differences.
Dose descriptor:
NOAEL
Remarks:
chronic effects
Effect level:
1.03 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male
Basis for effect level:
other: changes in the mesenteric lymph nodes of high dose animals: aggregates of macrophages often showing signs of degeneration and necrosis, in a few rats accompanied by disturbance of lymph node architecture
Dose descriptor:
NOAEL
Remarks:
chronic effects
Effect level:
1.38 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
female
Basis for effect level:
other: changes in the mesenteric lymph nodes of high dose animals: aggregates of macrophages often showing signs of degeneration and necrosis, in a few rats accompanied by disturbance of lymph node architecture
Dose descriptor:
NOAEL
Remarks:
chronic effects
Effect level:
100 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: changes in the mesenteric lymph nodes of high dose animals: aggregates of macrophages often showing signs of degeneration and necrosis, in a few rats accompanied by disturbance of lymph node architecture
Dose descriptor:
NOAEL
Remarks:
oncogenic effects
Effect level:
>= 3.27 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male
Basis for effect level:
other: No treatment-related neoplastic lesions were observed.
Dose descriptor:
NOAEL
Remarks:
oncogenic effects
Effect level:
>= 4.19 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
female
Basis for effect level:
other: No treatment-related neoplastic lesions were observed.
Dose descriptor:
NOAEL
Remarks:
oncogenic effects
Effect level:
>= 300 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment-related neoplastic lesions were observed.
Critical effects observed:
yes
Lowest effective dose / conc.:
3.27 mg/kg bw/day (actual dose received)
System:
immune system
Organ:
mesenteric lymph node
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
yes

Table A6.7-1:Results of the 2-year feeding study in rats.

Parameter

Control

30 ppm

100 ppm

300 ppm

Dose-response
+/–

 

m

f

m

f

m

f

m

f

m

f

Haematology

 

 

 

 

 

Packed cell volume (%)
week 13
week 26
week 52
week 78
week 102


47.4
48.6
47.4
44.9
45.9


44.4
46.2
43.6
43.9
43.0


47.3
48.1
46.1
45.1
44.2


45.0
45.6
45.3
43.3
42.4


47.2
46.8
46.4
46.3
44.8


46.1
47.1
45.7*
46.0*
44.4


45.3**
47.7
46.7
43.4
42.6**


46.7*
45.8
44.5
44.9
41.11)

 

 

Haemoglobin (g/100 mL)
week 13
week 26
week 52
week 78
week 102


15.9
16.0
15.3
14.9
14.1


14.8
15.2
14.8
14.3
13.2


15.8
15.7
15.1
15.0
13.9


15.1
15.3
14.9
14.5
13.5


16.2
15.5
15.2
15.1
13.7


15.3
15.5
15.5*
14.9*
13.8


15.4
15.8
14.9
14.1*
13.5*


15.4
15.3
14.9
14.5
12.81)

 

 

Clinical chemistry

 

 

 

 

 

 

 

 

 

 

Blood glucose (mg %)
week 26
week 52
week 104


67
79
76


84
78
81


68
76
74


84
77
73*


74*
82
80


85
74
75*


73
76
76


84
75
74*

 

 

Albumin (g %)
week 104


2.4


3.1


2.3


2.9


2.4


3.1


2.3


2.7*

 

 

Histopathological examinations: mesenteric lymph node alterations

Number examined

47

42

26

28

27

27

25

21

 

 

Grade 0

39

39

17

20

10

14

3

2

-

-

Grade 1

8

3

9

8

17

13

5

4

 

 

Grade 2

0

0

0

0

0

0

7

6

+

+

Grade 3

0

0

0

0

0

0

7

8

+

+

Grade 4

0

0

0

0

0

0

3

1

+

+

Moderate to marked erythrophagocytosis

2

0

2

0

0

0

0

1

 

 

Moderate no of macrophages containing brown pigment granules in their cytoplasm

0

3

2

0

0

0

0

0

 

 

Lymphoid hyperplasia

0

0

0

0

0

0

0

1

 

 

*) significantly different from control (p<0.05)
**) significantly different from control (p<0.01)
1) one female rat with very low values for Hb = 6.2, Hc = 21.0 and red blood cell counts = 2.6

Grade 0 = none / or only a few macrophages

Grade 1 = small number of macrophages

Grade 2 = moderate number of macrophages generally forming small aggregates

Grade 3 = medium-sized to large aggregates of macrophages, frequently showing signs of degeneration and necrosis, accompanied by proliferation of histiocytes

Grade 4 = large aggregates of histiocytes and macrophages, frequently showing necrotic areas. Marked reduction in size and number of lymphocytic follicle, or total disturbance of the normal lymph node architecture

 

Table A6.7-2:Test compound intake at relevant dietary concentrations.

 

 

Test compound intake (mg/kg bw/d)*

 

 

100 ppm

300 ppm

Parents

Male

3.8

12.1

 

Female

5.1

15.5

*) calculated with biweekly mean food intake values (week 1+2, 3+4, 11+12, 23+24, 35+36, 47+48, 59+60, 71+72, 83+84, 95+96) and weekly mean body weights (week 2, 4, 8, 12, 20, 28, 36, 44, 52, 64, 76, 88)

NOAEL/LOAEL = NOAEC/LOAEC × daily food consumption/body weight

 

Conclusions:
Based on the alterations in the mesenteric lymph nodes observed for both sexes, the chronic NOAEL was determined to be 100 ppm, corresponding to 1.03 mg a.i./kg bw/d for males and 1.38 mg a.i./kg bw/d for females.
Executive summary:

The chronic toxicity and carcinogenicity of Reaction product of lauryl-PDA/lauryl-DETA with chloroacetic acid (27% a.i.) was tested in Wistar rats. Thirty rats per sex per dose levels were fed the test substance at dietary concentrations of 30, 100 or 300 ppm for up to 2 years. A control group of 50 animals per sex received untreated diet. Although not a guideline study, the method used was consistent to EU method B.33 (88/303/EC) in all important aspects, with the following exceptions: 30 instead of 50 animals per sex and group were treated, no satellite groups were included, body weights were recorded biweekly instead of weekly during the first 12 weeks, food consumption was determined at intervals during periods of two weeks instead of weekly during the first 13 weeks and full histopathology was not carried out on all animals of the control and the high dose group.

No treatment-related effects on survival, general health and behaviour, body weight and food consumption were observed.

There were no clear treatment-related effects on clinical chemistry, urinalysis and organ weights. Decreased values for haemoglobin content and packed cell volume observed in high dose animals after 102 weeks of exposure were considered to be of minor toxicological relevance. Macroscopic examination did not indicate treatment-related lesions.

Histopathological examinations revealed treatment-related changes in the mesenteric lymph nodes of high dose animals only. These findings comprised aggregates of macrophages often showing signs of degeneration and necrosis, in a few rats accompanied by disturbance of lymph node architecture.

No treatment-related neoplastic lesions were observed.

 

Based on the alterations in the mesenteric lymph nodes observed for both sexes, the NOAEL (chronic effects) was determined to be 100 ppm, corresponding to 3.8 mg/kg bw/d for males and 5.1 mg/kg bw/d for females in term of test material (27% aqueous solution). In terms of active ingredient this corresponds to 1.03 mg a.i./kg bw/d for males and 1.38 mg a.i./kg bw/d for females.

 

The NOAEL for oncogenic effects was 300 ppm, corresponding to 12.1 mg/kg bw/d for males and 15.5 mg/kg bw/d for females in terms of test material (27% aqueous solution). In terms of active ingredient this corresponds to 3.27 mg a.i./kg bw/d for males and 4.19 mg a.i./kg bw/d for females.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
1.03 mg/kg bw/day
Study duration:
chronic
Species:
rat
System:
gastrointestinal tract
Organ:
mesenteric lymph node

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

For the assessment of repeated dose toxicity of Reaction product of lauryl-PDA/lauryl-DETA with chloroacetic acid a subchronic and a chronic toxicity study are available which were conducted with the target substance itself. A justification for read-across is attached to IUCLID section 13.

Subchronic toxicity studies

The toxicity of Reaction product of lauryl-PDA/lauryl-DETA with chloroacetic acid (27% aqueous solution) was examined in a sub-chronic (90-day) toxicity study with albino rats. The test material was fed at levels of 0, 0.01, 0.03, 0.1 and 0.3% in stock diet to groups of ten male and ten female weanling rats each. Observations were made of general appearance and behaviour, growth, food intake and efficiency, haematological factors and urine composition. Examinations were made on the blood serum for enzyme activities, viz, SGOT, SGPT, S.AP and for total serum protein and albumin.

At week 14 all rats were killed and examined grossly. Several organs were weighed and extensive histopathological examination was carried out.

At 0.3% growth depression, decreased food intake and food efficiency were observed in males. White blood cell counts were increased in both sexes at 0.3%. Differential counts showed a decrease of lymphocytes at 0.3% in females and abnormal looking lymphocytes in females at 0.03, 0.1 and 0.3%.

SGPT values were distinctly decreased at 0.3% in both sexes and at 0.1% in females. SAP activities were significantly decreased at the three highest feeding levels in females. Total serum protein and albumin content were decreased at 0.3% in both sexes and at 0.03 and 0.1% in males.

The relative weights of kidneys and spleen were increased at 0.3% in both sexes. Testicle weight was increased at 0.3%. At 0.1% kidney weight was increased in both sexes and spleen weight in males. At 0.03% all relative organ weights were within the normal range.

Gross and microscopic pathological examination revealed distinct treatment related changes in ileum and mesenteric lymph nodes consisting of accumulations of large foamy macrophages at 0.1% and 0.3% in both sexes.

It was concluded that the NOAEL of Reaction product of lauryl-PDA/lauryl-DETA with chloroacetic acid (27% aqueous solution) was 0.01% in the diet of rats for three months, which is equivalent to 5 mg/kg bw/d in terms of test material or 1.35 mg a.i./kg bw/d.

 

Additional data are available for the structurally related source substance DOPA-Glycinate.

The oral 90-day toxicity study was carried out according to EPA 82-1 (1982). Four groups of 10 male and 10 female Sprague Dawley rats, respectively, were administered orally by gavage 0, 25, 50 and 100 mg/kg bw/day of DOPA-Glycinate (20% aqueous solution, substance as manufactured), corresponding to 0, 5, 10 and 20 mg a.i./kg bw/d. for 90 days.

The conduct of the study was similar to OECD 408 (1998) with the exception that (i) no NOAEL could be determined, (ii) no sensory reactivity test, no assessment of grip strength, no motor activity assessment and no functional observations were performed, (iii) the weight variation at the commencement of the study exceeded ± 20 % for males, (iv) weights of epididymides, uterus, thymus, spleen, brain and heart were not determined, and (v) the mesenteric lymph nodes were not examined for the low and mid dose group despite observed effects at the high dose group.

Treatment with the test substance at a dose level of 100 mg/kg bw/day produced toxic effects manifested by foci of necrosis, acute inflammation and lymphoid hyperplasia of the mesenteric lymph nodes in animals of either sex. Since histopathological examination of mesenteric lymph nodes did not include the low dose and mid dose animals, the effect of the test substance at dose levels of 25 and 50 mg/kg bw/day on this tissue could not be clarified.

Further treatment-related findings were reduction in body weight gain and overall food consumption in male animals receiving 50 and 100 mg/kg bw/day, ophthalmological findings (congestion of the optic disk) in five high dose male animals and one female, haematological changes (significant increase of leucocytes and neutrophile granulocytes and decrease of lymphocytes) in males and females receiving 25, 50 and 100 mg/kg bw/day, slight blood chemistry changes (reduction in glucose, total protein, albumin, albumin/globulin ratio and cholesterol) within the normal range of background data in males and females treated with the test substance at 50 and 100 mg/kg bw/day, and a markedly increased alkaline phosphatase level in high dosed females only.

No treatment-related mortality or clinical signs occurred during the study.

Apart from slight leucocytosis, granulocytosis and lymphenia treatment with the test substance at a dose level of 25 mg/kg bw/day was very well tolerated and did not elicit any further toxic changes attributable to oral administration of the compound tested.

Based on a significant increase of leucocytes and neutrophile granulocytes and decrease of lymphocytes at all dose levels, the LOAEL was 25 mg/kg bw/d (as 20% aqueous solution), corresponding to 5 mg a.i./kg bw/d.

No NOAEL could be determined based on the results of this study. Therefore, a supplementary 90-day oral toxicity test utilising a modified dose range was conducted.

The supplementary oral 90-day toxicity study was carried out according to EPA 82-1 (1982). Three groups of 10 male and 10 female Sprague Dawley rats, respectively, were administered orally by gavage 0, 6.25 and 12.5 mg/kg bw/day of DOPA-Glycinate (20% aqueous solution, substance as manufactured) for 90 days.

The conduct of the study was consistent in all important aspects to OECD 408 (1998) with the exception that only 2 dose levels (6.25 and 12.50 mg/kg bw/day) instead of 3 were examined and that survival, general health, haematological parameters, gross necropsy and histopathology of mesenteric lymph nodes were investigated only. However, this study is a follow-up study to the above 90 d repeated dose toxicity study. Thus, the investigation of further parameters is not considered to be required.

No treatment-related clinical signs, mortalities, effects on food consumption and body weight, haematological changes, macroscopic lesions in any of the organs or tissues examined were observed during the study. Microscopic findings did not reveal any abnormalities of mesenteric lymph nodes.

Based on the results, the NOAEL in males and females was 12.50 mg/kg bw/d (as 20% aqueous solution), corresponding to 2.5 mg a.i./kg bw/d.

 

Chronic toxicity study

The chronic toxicity and carcinogenicity of Reaction product of lauryl-PDA/lauryl-DETA with chloroacetic acid (27% a.i.) was tested in Wistar rats. Thirty rats per sex per dose levels were fed the test substance at dietary concentrations of 30, 100 or 300 ppm for up to 2 years. A control group of 50 animals per sex received untreated diet. Although not a guideline study, the method used was consistent to EU method B.33 (88/303/EC) in all important aspects, with the following exceptions: 30 instead of 50 animals per sex and group were treated, no satellite groups were included, body weights were recorded biweekly instead of weekly during the first 12 weeks, food consumption was determined at intervals during periods of two weeks instead of weekly during the first 13 weeks and full histopathology was not carried out on all animals of the control and the high dose group.

No treatment-related effects on survival, general health and behaviour, body weight and food consumption were observed.

There were no clear treatment-related effects on clinical chemistry, urinalysis and organ weights. Decreased values for haemoglobin content and packed cell volume observed in high dose animals after 102 weeks of exposure were considered to be of minor toxicological relevance. Macroscopic examination did not indicate treatment-related lesions.

Histopathological examinations revealed treatment-related changes in the mesenteric lymph nodes of high dose animals only. These findings comprised aggregates of macrophages often showing signs of degeneration and necrosis, in a few rats accompanied by disturbance of lymph node architecture.

No treatment-related neoplastic lesions were observed.

Based on the alterations in the mesenteric lymph nodes observed for both sexes, the NOAEL (chronic effects) was determined to be 100 ppm, corresponding to 3.8 mg/kg bw/d for males and 5.1 mg/kg bw/d for females in term of test material (27% aqueous solution). In terms of active ingredient this corresponds to 1.03 mg a.i./kg bw/d for males and 1.38 mg a.i./kg bw/d for females.

The NOAEL for oncogenic effects was 300 ppm, corresponding to 12.1 mg/kg bw/d for males and 15.5 mg/kg bw/d for females in terms of test material (27% aqueous solution). In terms of active ingredient this corresponds to 3.27 mg a.i./kg bw/d for males and 4.19 mg a.i./kg bw/d for females.

 

Summary

In the 90 d repeated dose toxicity studies conducted with the target substance Reaction product of lauryl-PDA/lauryl-DETA with chloroacetic acid and the source substance DOPA-Glycinate as well as in the chronic toxicity study conducted with the target substance Reaction product of lauryl-PDA/lauryl-DETA with chloroacetic acid treatment related changes were observed in the mesenteric lymph nodes.

The target substance was slightly more toxic as demonstrated by the NOAEL obtained in the 90d study, which was lower by approx. a factor of 2. This is consistent with the observed effect levels in the acute oral toxicity studies.

 

The overall NOAEL for the target substance Reaction product of lauryl-PDA/lauryl-DETA with chloroacetic acid with respect to repeated dose toxicity is the NOAEL (males) of 1.03 mg a.i./kg bw/d obtained in the chronic toxicity study in rat.

 

There are no data gaps for the endpoint repeated dose toxicity. No human information is available for this endpoint. However, there is no reason to believe that these results would not be applicable to humans.

Justification for classification or non-classification

The classification for repeated dose toxicity is based on the findings of the subchronic and chronic studies in rat. Adverse effects were seen at dose levels <10 mg/kg bw/d. Thus, in accordance with CLP, EU GHS (Regulation 1272/2008/EC), Reaction product of lauryl-PDA/lauryl-DETA with chloroacetic acid is classified as STOT RE Cat 1, H372: Causes damage to organs (mesenteric lymph nodes) through prolonged or repeated exposure.