N-[3-(dimethoxymethylsilyl)propyl]ethylenediamine

Endpoint summary

Description of key information

In a 90-day repeated dose oral toxicity study with N-[3-(dimethoxymethylsilyl)propyl]ethylenediamine (CAS No. 3069-29-2, EC No. 221-336-6), conducted according to OECD Test Guideline 408 and in compliance with GLP, the NOAEL for systemic effects was concluded to be 300 mg/kg bw/day based on renal lesions observed at the highest dose tested, 500 mg/kg bw/day (BSL Bioservice, 2021).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27-07-2020 to 13-01-2021

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:

25 June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI(Han)

Details on species / strain selection:

Animal model: healthy Wistar rats, Crl: WI(Han) (Full Barrier)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River 97633 Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 5-6 weeks old
- Weight at study initiation: 185 g to 216 g (males); 135 g to 169 g (females)
- Fasting period before study: No
- Housing: The animals were kept in groups of 5 animals / sex / group / cage in IVC cages (type IV, polysulphone cages) on Altromin saw fibre bedding
- Diet: Altromin 1324 maintenance diet, ad libitum
- Water: Tap water, sulphur acidified to a pH of approximately 2.8, ad libitum
- Acclimation period: At least 5 days

DETAILS OF FOOD AND WATER QUALITY:
Municipal residue control and microbiological controls are done for the drinking water, at regular intervals.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3 °C
- Humidity (%): 55 +/-10%%
- Air changes (per hr): 10 times per hour
- Photoperiod: 12 hours light/12 hours dark

IN-LIFE DATES: From: July 2020 To: 01 December 2020

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was dissolved in dried and de-acidified corn oil. The test item was weighed into a tared glass vial on a suitable precision balance and the vehicle was added to give the appropriate final concentration of the test item. The formulation was alternately vortexed and/or stirred until visual homogeneity was achieved.
Based on the results of stability testing, the test item formulations were prepared fresh on each administration day before the administration procedure. The prepared formulations were stored protected from light and at room temperature. Formulates were kept under magnetic stirring during the daily administration. The vehicle was also used as the control item.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The vehicle was selected based on the test substance’s characteristics and the subsequent relevant OECD testing guidelines.
- Concentration in vehicle: 0, 25, 25, 75, 250/187.5/125* mg/mL
*The high dose level was reduced due to the irritating nature of the test substance and mortality at 1000 mg/kg bw/day starting on the 27th of August 2020, and at 750 mg/kg bw/day starting on the 11th of September 2020)
- Amount of vehicle (if gavage): 4 mL/kg body weight
- Lot/batch no. (if required): MKCK6411
- Purity: not specified

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples were collected for concentration analysis in weeks 1, 5, 9 and in the last week of treatment for all doses (16 samples in total). Each sample taken during the study was retained in duplicate (sample A, sample B, each of at least 3 mL). The A-samples were analysed at Eurofins Munich and until then stored under appropriate conditions based on available stability data. The B-samples are retained at below 15 °C at BSL Munich (test facility) and discarded after completion of the final study report.
Before beginning of the treatment period, formulation samples were prepared and analysed in order to obtain knowledge about stability and homogeneity of the test item in the selected vehicle at Eurofins Munich as part of a separate GLP study.

Duration of treatment / exposure:

90-day exposure

Frequency of treatment:

Once daily, 7 days per week

Dose / conc.:

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

Remarks:

Negative control group; Group 1

Dose / conc.:

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

Remarks:

Group 2

Dose / conc.:

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

Remarks:

Group 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

Group 4 [reduced to 750 and 500 mg/kg bw/day during the course of the study]

No. of animals per sex per dose:

10 animals per sex per dose. To determine reversibility of potential effects of the test item, additional recovery animals (5/sex in Group 4) were included in the study. Additional recovery animals (5/sex in Group 1 i.e. at dose level 0 mg/kg bw/day) were required for comparison purposes.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Doses for this study were selected based on the results of a 28-day dose range finding study (BSL Munich Study No STUGC19AA1217-1, non-GLP) in rats orally dosed daily at 50, 100, 250, 500 and 750 mg/kg bw/day for up to 4 weeks. Beginning on Day 15, females dosed at =600 mg/kg bw//day exhibited neurological clinical signs of abnormal gait, hindlimb splayed, impaired locomotion, flattened posture and/or decreased activity. There were no clinical signs, mortality, effects on food consumption, changes in haematological parameters, gross macroscopic lesions or toxicologically relevant differences in organ weights which could clearly be related to treatment with the test item up to 750 mg/kg bw/day. While there was a reduction in body weight gain in males at 500 (statistically significant) and in males and females at 750 (not statistically significant in either sex), overall the test item had no effect on body weight development (i.e. the mean body weight increased during the observation period in the control group and in all male and female treated groups). The clinical biochemistry parameters ALAT and ASAT were significantly increased in males at 750 mg/kg bw/day compared to the control group and may be dose-dependent. Based upon the absence of adverse findings in this dose range finding study and in keeping with the principle that “the highest dose level should be chosen with the aim to induce toxicity but not death or severe suffering”, the HD was increased in the subsequent main OECD 408 study to 1000 mg/kg bw/day. However, due to increased mortality in the HD group during this 90-day study, the HD was subsequently reduced from 1000 mg/kg bw/day to 750 mg/kg bw/day on 27 August 2020 (Day 24 of treatment: animal nos. 1-5, 11-15, 21-25, 31-35, Day 23 of treatment: animal nos. 6-10, 41-45, 91-95, 16-20, 26-30, 36-40, 46-50, 96-100, Day 22 of treatment: animal nos. 51-55, 61-65, 71-75, 81-85, Day 21 of treatment: animal nos. 56-60, 66-70, 76-80, 86-90) and then reduced again to 500 mg/kg bw/day on 11 September 2020 (Day 39 of treatment: animal nos. 1-5, 11-15, 21-25, 31-35, Day 38 of treatment: animal nos. 6-10, 41-45, 91-95, 16-20, 26-30, 36-40, 46-50, 96-100, Day 37 of treatment: animal nos. 51-55, 61-65, 71-75, 81-85, Day 36 of treatment: animal nos. 56-60, 66-70, 76-80, 86-90).


- Rationale for animal assignment (if not random): random

- Fasting period before blood sampling for clinical biochemistry: yes, animals were fasted overnight prior to each blood collection interval.

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations considering spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size were made outside the home cage in a standard arena once before the first administration and at least once a week thereafter.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: General clinical observations were made at least once a day, preferably at the same time each day and considering the peak period of anticipated effects after dosing. Inadvertently, the clinical observation was not recorded for the following animals on a single study day:
Day 9: animal no. 86
Day 10: animal nos. 81,83, 84, 85
Day 11: animal nos. 28, 40, 47, 96-99
Day 12: animal nos. 21,24,31, 34, 35


BODY WEIGHT: Yes
- Time schedule for examinations: Once before assignment to the experimental groups, on the first day of administration and weekly during the treatment and recovery periods.

FOOD CONSUMPTION AND COMPOUND INTAKE
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes. Food consumption was measured (weighed) weekly, during the treatment and recovery periods for each cage.


FOOD EFFICIENCY: No

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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: once before the first administration and in the last week of the treatment period, as well as at the end of the recovery period in the recovery animals. - Dose groups that were examined: all (surviving) animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at termination of dosing and at the end of recovery prior to or as part of the sacrifice of the animals
- Anaesthetic used for blood collection: Yes (ketamine/xylazin)
- Animals fasted: Yes
- How many animals: all surviving animals of the treatment/recovery period
- Parameters checked in table were examined. : yes, see Table 1

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination of dosing and at the end of recovery
- Animals fasted: Yes (ketamine/xylazin)
- How many animals: all suriviving animals of the treatment/recovery
- Parameters checked in table were examined: yes, see Table 1


URINALYSIS: Yes
- Time schedule for collection of urine: prior to or as part of the sacrifice of the animals
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Not specified
- Parameters examined: qualitative parameters: urine colour and appearance; quantitative indicators: specific gravity, nitrite, pH-value, protein, glucose


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once before the first exposure and once towards the end of exposure but not earlier than in week 11, as well as in the last week of the recovery period
- Dose groups that were examined: all animals
- Battery of functions tested: Functional observational battery (FOB) assessment was performed and the following parameters were observed: home cage evaluations (sleeps, piloerection, vocalizations, motor movements, grooming); examinations before transfer in open field/standard arena (salivation, lachrymation, changes in skin, cyanosis, exophthalmos, eyes opening, respiration, response to handling); examination immediately after transfer in open field (arousal, fear, finger approach, head touch); open field evaluations (body position, spontaneous locomotor activity, ataxic gait, hypotonic gait, twitches, tremors, seizures, unusual behaviour, stereotypical behaviour, rearing, urination, defaecation, faeces consistency, abnormal vocalisation, aggressiveness/irritability, grooming, startle response); examination while removing from open field and manipulating (equilibrium reflex, positional passivity, visual placing, grip strength, sensitivity to pinching the tail, toe pinch reflex, extensor thrust/limb tone, hind limb reflex, righting reflex on the ground, air righting reflex, pupil reflex, body temperature, anterior chamber of eye, fundus of eye)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see Table 2)

HISTOPATHOLOGY: Yes (see Table 2)

Statistics:

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 was performed for each gender by comparing values of treated animals with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests. Furthermore, statistical comparisons of data acquired during the recovery period was performed with a Student’s t-Test or Mann-Whitney U-Test when appropriate. These statistics were performed with Ascentos 1.3.4 software or GraphPad Prism V.6.01 software (p<0.05 is considered as statistically significant).

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no findings of toxicological relevance observed during the weekly detailed clinical observations during the treatment and recovery periods. No statistically significant differences were found between all treated groups and the respective control group.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Mortality was observed during the treatment period in the MD group (1/10 males, 2/10 females) and the HD group (9/15 males, 8/15 females), and additionally in one LD group female. No mortality occurred during the recovery period for the remaining HD group animals and the animals of the control group. Based on the histopathological evaluation, necrotic and/or inflammatory lesions were observed in the respiratory tract, as well as in the pleural region and/or oesophagus in decedents of the LD and HD groups, and were most likely involved in the animals’ death and morbidity. These lesions were considered to be due to incidental influx or gavage error during administration of the test item formulation with its irritating nature, and were deemed not to be due to a systemic effect of the test item. Furthermore, in decedents of the HD group, there were also necrotic and/or inflammatory lesions in the stomach. These stomach lesions were considered to be a local tissue injury associated with the irritating nature of the test item and were also likely to be involved in the animals’ death and morbidity.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Treatment with the test item showed no clear concentration-related statistically significant effect on body weight development in male and female animals of all treated groups. With the exception of a statistically significant increase in body weight of 40.86% in LD group males compared to the controls in the first week of the treatment period, there were no statistically significant differences in the male treated groups during the treatment and recovery periods and additionally, no statistically significant differences occurred during the treatment period in females. A single statistically significant reduction in body weight (6.65% below the control) was observed in the HD group females on day 118 during the recovery period and body weight change from day 97 to day 104 in these females was also reduced compared to the control (123.44% below the control); these changes were considered to be incidental and not related to treatment with the test item.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was no test item-related effect on food consumption of all male and female treated groups during the treatment and recovery periods.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The test item had no toxicologically relevant effect on haematological and coagulation parameters in male and female animals analysed at the end of the treatment and recovery periods.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The test item had no toxicologically relevant effect on clinical biochemical parameters and hormone analysis in male and female animals analysed at the end of the treatment and recovery periods.

Urinalysis findings:

no effects observed

Description (incidence and severity):

The test item had no toxicologically relevant effect on urinary parameters analysed in male and female animals at the end of the treatment and recovery periods.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

The statistically significant differences at the end of the treatment or recovery period were seen in only one gender, without dose dependency or only at the end of the recovery period. Therefore, no toxicological relevance was attributed to these observations.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The treatment with the test item showed an effect on the absolute and relative weight of the kidney to body weight and brain in the HD group, which was more prominent in males than in females. The absolute and relative (to body weight/brain) weight of kidney showed a statistically significant increase for HD group males, but the increase in HD females was lower than in males and showed no statistical significance. No statistical significance was noted for the differences in absolute and relative kidney weight compared to the control in LD and MD males and females. At the end of the recovery phase, the kidney weight recovered in HD males and the decrease in the female HD recovery group was not statistically significant below the control.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Histopathologically, most gross findings were considered to be a local tissue injury (due to incidental or accidental influx of the test item to the respiratory tract) associated with the irritating nature of the test item, or non-specific changes including agonal and postmortem changes that are frequently observed in dead or moribund animals. These findings included fluid filling or abnormal content in thoracic cavity, abnormal content in pericardium, alimentary tract filled with gas, abnormal colour and shape in several organs and tissues and failure to collapse in lungs. Thus, there were no gross lesions that could be due to a systemic effect of the test item in decedents.
The macroscopic findings which were observed for the surviving animals at scheduled necropsy were a dilated uterus in the control group and in several treated groups. Single findings in the LD group were white content in the urinary bladder and yellow fluid in the thoracic cavity. Based on the histopathological evaluation, there were no macroscopic findings in any survivors of the treated groups that could be attributed to treatment with the test item.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic changes that could be attributed to a systemic effect of the test item were observed in the kidney of both decedents and survivors. These included proximal tubular cell vacuolation, proximal tubular cell necrosis, and increased incidence and/or severity of tubular basophilia and tubular dilatation. These renal lesions were considered to correlate with a significant increase or increased tendency in kidney weights of the HD group. In animals necropsied at the end of the recovery period, proximal tubular vacuolation and necrosis were not found in any HD animals, and there were no qualitative differences in the other renal findings between the control and HD groups. There were also no differences in kidney weights in both sexes. Thus, the histopathological examination revealed that the treatment- related renal changes completely subsided during the recovery phase of this study.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

no

Relevant for humans:

not specified

Conclusions:

In a 90-day oral repeated dose toxicity study with the registered substance N-[3-(dimethoxymethylsilyl)-propyl]ethylenediamine, conducted according to OECD Test Guideline 408 and in compliance with GLP, the NOAEL for systemic effects was concluded to be 300 mg/kg bw/day based on renal lesions.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subchronic

Species:

rat

System:

urinary

Organ:

kidney

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

23 February 2017 to December 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

not specified

Remarks:

There are no deviations from the test guideline that affect the integrity of the study

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In a room with controls set to maintain 18°C to 24°C and under dry nitrogen gas, keep away from moisture with dessicant.
- Stability under test conditions: The test article is considered to be stable.
- Solubility and stability of the test substance in the solvent/vehicle: Filtered air was used as vehicle.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: not applicable
- Preliminary purification step (if any): not applicable
- Final dilution of a dissolved solid, stock liquid or gel: not applicable
- Final preparation of a solid: not applicable

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approximately 7 weeks
- Weight at study initiation: 227 to 307 g (males)/149 to 192 g (females)
- Fasting period before study: Food and water were not available during the exposure period.
- Housing: All animals were housed in groups of 2 to 4 per cage following receipt in clean, solid bottom cages with bedding material.
- Diet: PMI Nutrition International, LLC Certified Rodent LabDiet® 5002 meal was available ad libitum
- Water: Reverse osmosis-treated water was available ad libitum
- Acclimation period: 14 days

DETAILS OF FOOD AND WATER QUALITY: Filters servicing the automatic watering system were changed regularly according to SOPs. The municipal water supplying the laboratory was analyzed for contaminants according to SOPs. SOPs provide specifications for acceptable levels of heavy metals and pesticides that are reasonably expected to be present in the diet without interfering with the purpose or conduct of the study. No contaminants were reasonably expected to be present in water or diet that would interfere with the objectives of the study, therefore, no testing was conducted as part of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 20°C and 26°C
- Humidity (%): 15-30%
- Air changes (per hr): 10 per hour
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

> 1.3 - <= 2.1 µm

Geometric standard deviation (GSD):

2.47

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: stainless steel, conventional nose-only exposure systems
- Method of holding animals in test chamber: nose-only exposure restraint tubes
- Source and rate of air:
- Method of conditioning air:
- System of generating particulates/aerosols: Liquid droplet aerosol atmospheres of the test substance were generated using single-jet Collision nebulizers. Test substance exposure atmospheres were generated by mixing the aerosolized test substance with nitrogen and/or breathing-quality dry, compressed air in the appropriate ratio to achieve the target concentrations. Specific exposure atmosphere generation equipment, operating parameters and methods were defined during method development.
- Temperature, humidity, pressure in air chamber: Average daily temperature and relative humidity of the exposure atmospheres were 23 ± 3ºC and 15%-30%, respectively. Temperature and relative humidity were monitored continuously and recorded at approximately 60-minute intervals during each exposure.
- Air flow rate: Airflow rate through the system was set based on output from the generation equipment and the dilution airflow to provide sufficient volumes for the number of animals to be exposed and for test atmosphere sampling.
- Air change rate: 10 per hour
- Method of particle size determination: Aerosol particle size measurements were conducted during method development as needed and at least twice weekly during the 13-week exposure phase for all test substance exposure groups. Aerosol particle size measurements were conducted using a 7-stage stainless steel cascade impactor. The aerosol size was expressed in terms of the mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD). The target range for MMAD is 1.0 to 3.0 microns and GSD is 1.5 to 3.5. The mean aerosol particle size exposure system 2, 3 and 4 were 2.1, 1.9 and 1.3 microns, respectively. Mean GSD for exposure system 2, 3 and 4 were 2.35, 2.47 and 2.15, respectively.
- Treatment of exhaust air: exhaust passed through a Solberg canister filter prior to entering the facility exhaust system, which consists of redundant exhaust blowers preceded by activated charcoal- and HEPA-filtration units.

TEST ATMOSPHERE
- Brief description of analytical method used: gravimetric
- Samples taken from breathing zone: yes

VEHICLE (if applicable)
- Justification for use and choice of vehicle: filtered air
- Composition of vehicle: not applicable
- Type and concentration of dispersant aid (if powder): not applicable
- Concentration of test material in vehicle: not applicable
- Lot/batch no. of vehicle (if required): not applicable
- Purity of vehicle: not applicable

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples were collected using pre-weighed filters held in a filter holder and placed in the animal breathing zone of an exposure system. A volume of the test atmosphere is pulled through the filter. Following sample collection, the sample is reweighed. The exposure concentration (mg/m3) were calculated by dividing the gravimetrically determined mass of test substance aerosol by the sample volume. The mass was determined by subtracting the initial filter weight from the final weight of the post-sample filter. Sample volume was calculated by multiplying the sample flow rate by the length of the sampling period. Concentrations were not be adjusted for test substance purity. For the test substance exposure systems, samples were collected daily at appropriate intervals based on the target exposure concentrations. For the control group (Group 1), gravimetric samples were collected at least twice weekly during the 13-week exposure period.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

5 days a week for 6 hours a day

Dose / conc.:

5 mg/m³ air (nominal)

Dose / conc.:

15 mg/m³ air (nominal)

Dose / conc.:

45 mg/m³ air (nominal)

Dose / conc.:

5.1 mg/m³ air (analytical)

Dose / conc.:

14.9 mg/m³ air (analytical)

Dose / conc.:

44 mg/m³ air (analytical)

No. of animals per sex per dose:

20

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Exposure concentrations were selected to cover a range extending from an anticipated no observed-adverse-effect-concentration (NOAEC) to a concentration in which toxic effects are seen in the highest exposure concentration. Specific concentrations were based on the results of a 28-day range-finding study in which microscopic findings were noted in the lungs, trachea larynx and nasal levels at a concentration of 100 mg/m3.
- Rationale for animal assignment (if not random): random
- Post-exposure recovery period in satellite groups: The animals scheduled for the recovery evaluations will be subjected to necropsy following a 4-week non-exposure period (minimum of 27 days).

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed twice daily, once in the morning and once in the afternoon. Moribund animals were euthanized and necropsied as soon as possible. Animals found dead were necropsied as soon as possible to ensure that tissues will not be lost due to autolysis.
- Cage side observations checked in table were included: mortality, abnormalities, and signs of pain and distress

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Prior to treatment, weekly during the study period and on the day of necropsy

BODY WEIGHT: Yes
- Time schedule: Prior to treatment, twice weekly during the study period and on the day of necropsy

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 / No / Not specified

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule: Once during acclimation prior to randomization and all surviving animals near the end of the exposure period or near the end of the recovery period.
- Dose groups that were examined: all animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to necropsy
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to necropsy
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table [No.1] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: on the day of necropsy
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table [No.1] were examined.

NEUROBEHAVIOURAL EXAMINATION: No
- Time schedule for examinations: not conducted, as not triggered by clinical observations

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table No 2)
HISTOPATHOLOGY: Yes (see table No 2)

Statistics:

All analyses were two-tailed for significance levels of 5% and 1%. All means were presented with standard deviations. All statistical tests were performed by a computer with appropriate programming as referenced below.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were no clinical observations attributable to treatment in the low and mid-dose groups, or in the recovery groups. There were no clinical signs noted 0-1 hour following exposure in any group.

The only group with clinical signs of toxicity due to treatment was the high dose (45 mg/m3) group during the treatment period. Males and females of this group had multiple instances of red, brown, and/or yellow material on various body surfaces and clear discharge around the eyes. The staining was judged by the study author to be indicative of test substance-related stress or general ill health. This finding was observed prior to dosing and on non-exposure days during the treatment period.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One male in the 15 mg/m3 group was euthanized in extremis on Study Day 61 due to a clinical observation of red penile discharge. At necropsy, this male was noted with gross observations of dilated pelvis, distended bladder, distended ureters, and red fluid in the bladder and ureters. This death was not considered to be test substance-related. In addition, a single male in the control group was found dead on Study Day 65; there were no remarkable clinical observations or gross observations noted for this animal. All other animals survived to the scheduled necropsies.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There were no treatment-related effects on body weight or body weight gain in the low and mid-dose groups.

Test substance-related effects on body weights were noted in the 45 mg/m3 group males and females. Lower mean body weights and cumulative body weight gains were noted beginning as early as Study Week 1 in males and females, which continued until the primary necropsy (Study Week 13); the differences were generally significant compared to the control group. On Study Week 13 (the day of the primary necropsy), mean body weights in the 45 mg/m3 males and females were 9.7% and 6.4% lower, respectively, than the mean control group body weights. In addition, body weight gains in the 45 mg/m3 group males and females were comparable or higher than the control group values during the recovery period; while mean body weights in the males remained lower (not statistically significant) than the control group. Therefore, the animals recovered once exposure stopped, but males were slightly slower to recover than females.

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:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The only treatment-related effect on organ weights occurred in the high dose group and was limited to the lungs. There were no adverse findings in the low and mid-dose groups.

At the end of the treatment period: Lung weights (absolute and relative to body and brain weights) tended to be higher with exposure concentration across all groups with statistical significance achieved only at 45 mg/m3. For the males, all group mean and individual animal absolute lung weights, as examples, were within historical control reference ranges. In contrast for the 45 mg/m3 group females, mean absolute and both mean relative weights exceeded their historical control reference ranges and, as an example, 6 individual animal absolute lung weights also exceeded the historical control reference range. The most likely microscopic correlations for the higher lung weights were the findings affecting the bronchioles (epithelial hyperplasia, mixed cell infiltrates and luminal debris). Since none of these findings were seen in either sex at exposure concentrations < 45 mg/m3, the 8% to 10% higher lung weights seen in the 15 mg/m3 group males (and lesser magnitudes of change seen at lower exposure concentrations in both sexes), which were reversible, were considered incidental and unrelated to treatment.

At the end of the recovery period: treatment-related and statistically significant higher lung weights (absolute and/or relative to body and brain weights) persisted in both sexes at exposure concentrations of 45 mg/m3. Although mean absolute lung weight was not statistically significant in the 45 mg/m3 group males, the approximately 10% increase was considered sufficient to be considered an effect of test substance exposure. Peribronchiolar chronic inflammation was considered the microscopic correlate for the persistent high lung weights in this group.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Macroscopic findings were observed in the high dose group only at the end of the treatment period and were limited to the lungs. There were no adverse findings in any of the groups at the end of the recovery period.

Treatment-related findings of not fully collapsed (3/10 males and 8/10 females) and pale lungs (1/10 males and 2/10 females) were observed exclusively in the 45 mg/m3 group at the primary necropsy; luminal debris in the bronchioles and peribronchiolar inflammatory cell infiltrates appeared to be the microscopic correlates for lungs not fully collapsed while a correlation was not observed for pale lungs. At the Study Week 17 recovery necropsy, not fully collapsed lungs
was seen in 2 males and 1 female from the 45 mg/m3 group but also 1 male each from the control and 5 mg/m3 groups. Therefore at the recovery necropsy, lungs not fully collapsed was not conclusively associated with exposure to the test substance aerosol.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

See Tables 3 to 6.
Treatment-related microscopic findings were limited to the respiratory tract. These findings were observed in all groups at the end of the treatment period, but they were only observed in the high dose group at the end of the recovery period.

At the end of the treatment period: In the nasal cavity treatment-related epithelial (transitional, respiratory, or olfactory) degeneration/regeneration was characterized by loss of normal tissue architecture, most commonly by cell loss and replacement by a single layer of attenuated epithelium; these changes were typically bilateral. Specific to the respiratory epithelium was loss of cilia and reduction/loss of goblet cells. The sites of respiratory epithelial degeneration/regeneration were most commonly the dorsal aspect of the nasal septum and lining the dorsal meatus, including nasal turbinate(s). Olfactory epithelial degeneration/regeneration was limited to 1 female in the 45 mg/m3 group and was diffuse within the dorsal meatus of nasal level III while focal and unilateral in the posterior nasal levels. Transitional epithelial degeneration/regeneration was most commonly limited to the tissues lining the lateral meatus (nasal turbinates and lateral wall). Squamous cell metaplasia and mixed cell inflammation were usually associated with areas of epithelial degeneration/regeneration. Squamous cell metaplasia represented a change from the normal epithelium lining a region to an attenuated stratified and keratinized squamous epithelium. Mixed cell inflammation was characterized by a mononuclear cell (lymphocytes, plasma cells and macrophages) population admixed with variable numbers of neutrophils. In contrast to epithelial degeneration/regeneration seen most commonly in the females, transitional and respiratory epithelial hyperplasia was the most common epithelial change seen in the males exposed to test substance aerosol; affected regions were comparable to those seen with degeneration/regeneration in the females. Several of these findings (transitional epithelial hyperplasia, mixed cell inflammation, and squamous cell metaplasia) at nasal level II were seen in one or both sexes at exposure concentrations of 5, 15, and 45 mg/m3.

In the larynx, trachea, and bronchi (recorded under lungs) treatment-related respiratory epithelial degeneration/regeneration and mixed cell inflammation were comparable to that seen in the nasal cavity. It was not uncommon in control group animals to see respiratory epithelial loss and/or attenuation at these sites. Loss was clearly an artifact of tissue collection/processing while the cause of the thinned epithelium was less certain. However, respiratory epithelial degeneration/regeneration was diagnosed only when there was single cell necrosis, mitotic figure(s) and/or rare neutrophils (sub- or intra-epithelial) associated with the attenuated epithelium. Disruption of the normal epithelial architecture with loss of cilia was also considered degeneration/regeneration. In the larynx, squamous metaplasia was similar to that
seen in the nasal cavity except that it was more commonly seen as a change from the normal non-keratinized stratified epithelium to an attenuated and keratinized stratified squamous epithelium. In addition to mixed cell inflammation in the larynx, chronic active inflammation was also an effect of exposure to the test substance, particularly in the 45 mg/m3 group females, and the epiglottis was a common site of this inflammation. Chronic active inflammation was similar to mixed cell inflammation except in the former, fibroblasts/fibroplasia were present generally indicating inflammation of longer duration. Several of these findings (respiratory epithelial
degeneration of bronchi and mixed cell inflammation of the larynx and bronchi) were seen in one or both sexes at exposure concentrations of 5, 15, and 45 mg/m3.

At the end of the recovery period: In the nasal cavity (levels II and III) most previous effects of exposure persisted in one or both sexes from the 45 mg/m3 group with
incidences and severities that were generally lower than those seen at the primary necropsy. A new effect, and evidence of chronic irritation, was mucous cell hyperplasia of the nasal septum at nasal level II in the males. Due to the low incidence and minimal to mild severity and resolution during the 4-week recovery period, findings in the nasal cavity were considered to be nonadverse at 5 and 15 mg/m3.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Details on results:

In the lungs at the primary necropsy, treatment-related effects were centered on the bronchi and/or bronchioles. Changes in the bronchi were comparable to and considered an extension of the changes seen in the trachea. The most prominent change was noted at the periphery of the lungs at the level of the terminal bronchioles. In control group animals, terminal bronchioles rarely had any associated inflammatory or mononuclear cell infiltrates and the epithelial lining
was an admixture of ciliated respiratory epithelial cells and cells with distinct apical blebbing (club/Clara cells). Exposure-related effects included variably dense perivascular and/or peribronchiolar mixed cell infiltrates characterized by mononuclear cells with generally fewer eosinophils and/or neutrophils and in some cases the suggestion of increased fibrosis/fibroplasia (Masson’s stain suggested this was true in a 45 mg/m3 group female evaluated). There was loss of the normal epithelium lining the terminal bronchioles with replacement by a prominent respiratory epithelium with a robustly ciliated epithelium; club cells were generally absent (mild respiratory epithelial hyperplasia). With minimal respiratory epithelial hyperplasia, club cells were occasionally present and/or there was epithelial alteration comparable to the respiratory epithelial degeneration/regeneration noted in the bronchi. Some animals had bronchiolar luminal debris (exudate) consisting of a mucinous material admixed with foamy alveolar macrophages and less commonly neutrophils. It was this exudate in combination with the peribronchiolar cellular infiltrates and respiratory epithelial hyperplasia that were the most likely microscopic correlations for the lungs noted at necropsy as not fully collapsed. Other than the changes affecting the bronchi, discussed above, all treatment-related effects to the lower airways were limited to the 45 mg/m3 group.

In the lungs at the recovery necropsy, some of the previous effects of exposure persisted in one or both sexes from the 45 mg/m3 group with incidences and severities that were generally lower than those seen at the primary necropsy. A new effect, and evidence of chronic irritation, was the shift from mixed cell peribronchiolar infiltrates to chronic inflammation of the bronchioles. Chronic inflammation was characterized by peribronchiolar fibrosis admixed with mononuclear cell infiltrates. Although the absence of club cells was a component of respiratory epithelial hyperplasia of the bronchioles at Study Week 13, they remained reduced in number to absent at the recovery necropsy. Aggregates of pigmented alveolar macrophages, most likely containing hemosiderin, was also a new apparent effect of test substance administration in the 45 mg/m3 group males. Two males from the 45 mg/m3 group had proliferative changes characterized as ‘atypical’ including bronchiolo-alveolar hyperplasia and bronchial mucous cell metaplasia.

Key result

Dose descriptor:

NOAEC

Effect level:

ca. 15 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

45 mg/m³ air (nominal)

System:

other: whole respiratory tract

Organ:

larynx

lungs

nasal cavity

trachea

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Table 3 Incidence of Selected Histopathologic Findings, Study Week 13 Primary Necropsy - males

	Males
Exposure Concentration (mg/m^3)	0	5	15	45
Nasal level II^a	11^b	10	11^b	10
Degeneration/regeneration, respiratory epithelium	0	0	0	2
Minimal	-	-	-	2
Mild	-	-	-	0
Moderate	-	-	-	0
Degeneration/regeneration, transitional epithelium	0	0	0	0
Minimal	-	-	-	-
Mild	-	-	-	-
Moderate	-	-	-	-
Marked	-	-	-	-
Hyperplasia, respiratory epithelium	0	0	0	6
Minimal	-	-	-	4
Mild	-	-	-	2
Hyperplasia, transitional epithelium	1	4	6^b	3
Minimal	1	4	6^b	2
Mild	0	0	0	1
Inflammation, mixed cell	0	2	5	10
Minimal	-	2	4	10
Mild	-	0	1	0
Metaplasia, squamous cell	0	1	4	6
Minimal	-	1	4	4
Mild	-	0	0	2
Nasal level III^a	11^b	NA	NA	10
Degeneration/regeneration, olfactory epithelium (marked)	0	-	-	0
Degeneration/regeneration, respiratory epithelium	0	-	-	1
Minimal	-	-	-	1
Mild	-	-	-	0
Larynx^a	11^b	10	10^b	10
Degeneration/regeneration, respiratory epithelium(minimal)	0	0	1	4
Inflammation, chronic active	0	0	2	1
Minimal	-	-	0	0
Mild	-	-	2	1
Inflammation, mixed cell	2	2	5^b	6
Minimal	2	2	3^b	4
Mild	0	0	2	2
Metaplasia, squamous cell	0	0	0	2
Minimal	-	-	-	1
Mild	-	-	-	1
Trachea^a	11^b	9	11^b	10
Degeneration/regeneration, respiratory epithelium	0	0	3^b	7
Minimal	-	-	2^b	5
Mild	-	-	1	2
Moderate	-	-	0	0
Inflammation, mixed cell (minimal)	1^b	0	1	3
Lungs^a	11^b	10	11^b	10
Degeneration/regeneration, respiratory epithelium, bronchus	0	4	9^b	7
Minimal	-	4	9^b	5
Mild	-	0	0	2
Hyperplasia, respiratory epithelium, bronchioles	0	0	0	7
Minimal	-	-	-	6
Mild	-	-	-	1
Infiltrate, mixed cell, peribronchiolar	0	0	0	7
Minimal	-	-	-	3
Mild	-	-	-	4
Inflammation, mixed cell, bronchus	0	3	3	4
Minimal	-	3	3	3
Mild	-	0	0	1
Luminal debris, bronchioles	0	0	0	2
Minimal	-	-	-	0
Mild	-	-	-	2

^a Number of tissues examined from each group.

^b Includes 1 FD/EE rat.

BOLDED incidences represent effects of exposure to N-[3-(trimethoxysilyl)propyl]ethylenediamine, CAS 1760-24-3

Table 4 Incidence of Selected Histopathologic Findings, Study Week 13 Primary Necropsy - females

	Females
Exposure Concentration (mg/m^3)	0	5	15	45
Nasal level II^a	10	10	10	10
Degeneration/regeneration, respiratory epithelium	0	0	0	8
Minimal	-	-	-	2
Mild	-	-	-	2
Moderate	-	-	-	4
Degeneration/regeneration, transitional epithelium	0	0	1	8
Minimal	-	-	1	1
Mild	-	-	0	1
Moderate	-	-	0	2
Marked	-	-	0	4
Hyperplasia, respiratory epithelium	0	0	0	0
Minimal	-	-	-	-
Mild	-	-	-	-
Hyperplasia, transitional epithelium	0	0	0	0
Minimal	-	-	-	-
Mild	-	-	-	-
Inflammation, mixed cell	2	5	4	8
Minimal	2	3	4	3
Mild	0	2	0	5
Metaplasia, squamous cell	0	0	1	4
Minimal	-	-	0	4
Mild	-	-	1	0
Nasal level III^a	10	10	10	10
Degeneration/regeneration, olfactory epithelium (marked)	0	0	0	1
Degeneration/regeneration, respiratory epithelium	0	0	0	3
Minimal	-	-	-	1
Mild	-	-	-	2
Larynx^a	10	10	10	10
Degeneration/regeneration, respiratory epithelium(minimal)	0	0	1	1
Inflammation, chronic active	0	0	0	5
Minimal	-	-	-	2
Mild	-	-	-	3
Inflammation, mixed cell	1	4	7	7
Minimal	1	4	5	6
Mild	0	0	2	1
Metaplasia, squamous cell	0	0	0	5
Minimal	-	-	-	5
Mild	-	-	-	0
Trachea^a	10	10	10	10
Degeneration/regeneration, respiratory epithelium	0	0	2	8
Minimal	-	-	1	3
Mild	-	-	1	4
Moderate	-	-	0	1
Inflammation, mixed cell (minimal)	0	0	1	5
Lungs^a	10	10	10	10
Degeneration/regeneration, respiratory epithelium, bronchus	0	1	5	9
Minimal	-	1	5	7
Mild	-	0	0	2
Hyperplasia, respiratory epithelium, bronchioles	0	0	0	10
Minimal	-	-	-	10
Mild	-	-	-	0
Infiltrate, mixed cell, peribronchiolar	0	0	0	10
Minimal	-	-	-	5
Mild	-	-	-	5
Inflammation, mixed cell, bronchus	0	1	0	6
Minimal	-	1	-	6
Mild	-	0	-	0
Luminal debris, bronchioles	0	0	0	7
Minimal	-	-	-	3
Mild	-	-	-	4

^a Number of tissues examined from each group.

^b Includes 1 FD/EE rat.

BOLDEDincidences represent effects of exposure to N-[3-(trimethoxysilyl)propyl]ethylenediamine, CAS 1760-24-3

Table 5 Incidence of Selected Histopathologic Findings, Study Week 17 Recovery Necropsy - males

	Males
Exposure Concentration (mg/m^3)	0	5	15	45
Nasal level II^a	9	10	9	10
Degeneration/regeneration, respiratory epithelium(minimal)	0	0	0	1
Degeneration/regeneration, transitional epithelium	0	0	0	1
Minimal	-	-	-	1
Mild	-	-	-	0
Hyperplasia, mucous cell (minimal)	0	1	1	5
Hyperplasia, respiratory epithelium (mild)	0	0	0	2
Inflammation, mixed cell	2	1	1	3
Minimal	2	1	1	1
Mild	0	0	0	2
Nasal level III^a	NA	NA	NA	NA
Degeneration/regeneration, olfactory epithelium (moderate)	-	-	-	-
Inflammation, mixed cell (mild)	-	-	-	-
Larynx^a	9	10	9	10
Degeneration/regeneration, respiratory epithelium(minimal)	0	0	0	0
Inflammation, chronic	0	0	0	4
Minimal	-	-	-	1
Mild	-	-	-	3
Trachea^a	9	10	9	10
Degeneration/regeneration, respiratory epithelium (Minimal)	0	0	0	0
Inflammation, mixed cell (minimal)	0	0	0	0
Lungs^a	9	10	9	10
Decreased club cells, terminal bronchioles (Present)	0	0	0	8
Degeneration/regeneration, respiratory epithelium, bronchus (Minimal)	0	0	0	0
Inflammation, chronic, bronchioles (Minimal)	0	0	0	7
Luminal debris, bronchioles (Minimal)	0	0	0	2
Macrophages, pigmented (Minimal)	0	1	0	3

^a Number of tissues examined from each group.

BOLDEDincidences represent effects of exposure to N-[3-(trimethoxysilyl)propyl]ethylenediamine,

CAS 1760-24-3

There were no other test substance-related histologic changes. Remaining histologic changes were considered to be incidental findings or related to some aspect of experimental manipulation other than exposure to N-[3-(trimethoxysilyl)propyl]ethylenediamine. There was no test substance-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Table 6 Incidence of Selected Histopathologic Findings, Study Week 17 Recovery Necropsy - females

	Females
Exposure Concentration (mg/m^3)	0	5	15	45
Nasal level II^a	10	10	10	10
Degeneration/regeneration, respiratory epithelium(minimal)	0	0	0	1
Degeneration/regeneration, transitional epithelium	0	0	0	2
Minimal	-	-	-	0
Mild	-	-	-	2
Hyperplasia, mucous cell (minimal)	0	0	0	0
Hyperplasia, respiratory epithelium (mild)	0	0	0	0
Inflammation, mixed cell	2	0	1	5
Minimal	2	-	1	4
Mild	0	-	0	1
Nasal level III^a	10	10	10	10
Degeneration/regeneration, olfactory epithelium (moderate)	0	0	0	1
Inflammation, mixed cell (mild)	0	0	0	1
Larynx^a	10	10	10	10
Degeneration/regeneration, respiratory epithelium(minimal)	0	0	0	3
Inflammation, chronic	0	0	0	6
Minimal	-	-	-	3
Mild	-	-	-	3
Trachea^a	10	10	10	10
Degeneration/regeneration, respiratory epithelium (Minimal)	0	0	0	1
Inflammation, mixed cell (minimal)	0	0	0	1
Lungs^a	10	10	10	10
Decreased club cells, terminal bronchioles (Present)	0	0	0	10
Degeneration/regeneration, respiratory epithelium, bronchus (Minimal)	0	0	0	1
Inflammation, chronic, bronchioles (Minimal)	0	0	0	9
Luminal debris, bronchioles (Minimal)	0	0	0	5
Macrophages, pigmented (Minimal)	0	0	0	0

^a Number of tissues examined from each group.

BOLDEDincidences represent effects of exposure to N-[3-(trimethoxysilyl)propyl]ethylenediamine,

CAS 1760-24-3

There were no other test substance-related histologic changes. Remaining histologic changes were considered to be incidental findings or related to some aspect of experimental manipulation other than exposure to N-[3-(trimethoxysilyl)propyl]ethylenediamine. There was no test substance-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Table 7 Correlation of Selected Observations

 

Necropsy	Organ Weight	Clinical Pathology	Histopathology
Lung- not fully collapsed (PN; 45 mg/m^3)	¿ lung weight	-	Lung - Luminal debris, bronchioles; respiratory epithelial hyperplasia of the bronchioles and mixed cell infiltrate, peribronchiolar
-	¿ lung weight (RN;  45 mg/m^3)	-	Lung- Luminal debris, bronchioles and chronic inflammation, bronchioles

 

 Relationships were suspected between gross necropsy, organ weight, clinical pathology, and histopathology observations, as presented in Table 7. These proposed relationships were based on subjective interpretation rather than a statistical analysis of correlation.

Conclusions:

In a 90-day repeated dose toxicity study conducted according to OECD Test Guideline 413 and in compliance with GLP, exposure of Sprague-Dawley rats to N-[3-(trimethoxysilyl)propyl] ethylenediamine via nose-only inhalation for 6 hours per day on a 5-day basis for 13 weeks at target exposure concentrations of 5, 15, and 45 mg/m3 resulted in non-adverse test substance-related effects at 5 and 15 mg/m3. Adverse test substance-related clinical observations, lower body weights and body weight gains, macroscopic findings in the lungs, increased lung weights, and microscopic findings in the nasal cavity, larynx, trachea, and lungs were noted in the 45 mg/m3 group males and females at the end of the treatment period. At the recovery necropsy, many of the effects of test substance exposure, including higher lung weights, persisted in the nasal cavity, larynx, trachea, and lungs in the 45 mg/m3 group and were noted at generally a lower incidence and severity than that seen at the primary necropsy. Therefore, the NOAEC was concluded to be 15 mg/m3.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

23 February 2017 to December 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

not specified

Remarks:

There are no deviations from the test guideline that affect the integrity of the study

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In a room with controls set to maintain 18°C to 24°C and under dry nitrogen gas, keep away from moisture with dessicant.
- Stability under test conditions: The test article is considered to be stable.
- Solubility and stability of the test substance in the solvent/vehicle: Filtered air was used as vehicle.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: not applicable
- Preliminary purification step (if any): not applicable
- Final dilution of a dissolved solid, stock liquid or gel: not applicable
- Final preparation of a solid: not applicable

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approximately 7 weeks
- Weight at study initiation: 227 to 307 g (males)/149 to 192 g (females)
- Fasting period before study: Food and water were not available during the exposure period.
- Housing: All animals were housed in groups of 2 to 4 per cage following receipt in clean, solid bottom cages with bedding material.
- Diet: PMI Nutrition International, LLC Certified Rodent LabDiet® 5002 meal was available ad libitum
- Water: Reverse osmosis-treated water was available ad libitum
- Acclimation period: 14 days

DETAILS OF FOOD AND WATER QUALITY: Filters servicing the automatic watering system were changed regularly according to SOPs. The municipal water supplying the laboratory was analyzed for contaminants according to SOPs. SOPs provide specifications for acceptable levels of heavy metals and pesticides that are reasonably expected to be present in the diet without interfering with the purpose or conduct of the study. No contaminants were reasonably expected to be present in water or diet that would interfere with the objectives of the study, therefore, no testing was conducted as part of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 20°C and 26°C
- Humidity (%): 15-30%
- Air changes (per hr): 10 per hour
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

> 1.3 - <= 2.1 µm

Geometric standard deviation (GSD):

2.47

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: stainless steel, conventional nose-only exposure systems
- Method of holding animals in test chamber: nose-only exposure restraint tubes
- Source and rate of air:
- Method of conditioning air:
- System of generating particulates/aerosols: Liquid droplet aerosol atmospheres of the test substance were generated using single-jet Collision nebulizers. Test substance exposure atmospheres were generated by mixing the aerosolized test substance with nitrogen and/or breathing-quality dry, compressed air in the appropriate ratio to achieve the target concentrations. Specific exposure atmosphere generation equipment, operating parameters and methods were defined during method development.
- Temperature, humidity, pressure in air chamber: Average daily temperature and relative humidity of the exposure atmospheres were 23 ± 3ºC and 15%-30%, respectively. Temperature and relative humidity were monitored continuously and recorded at approximately 60-minute intervals during each exposure.
- Air flow rate: Airflow rate through the system was set based on output from the generation equipment and the dilution airflow to provide sufficient volumes for the number of animals to be exposed and for test atmosphere sampling.
- Air change rate: 10 per hour
- Method of particle size determination: Aerosol particle size measurements were conducted during method development as needed and at least twice weekly during the 13-week exposure phase for all test substance exposure groups. Aerosol particle size measurements were conducted using a 7-stage stainless steel cascade impactor. The aerosol size was expressed in terms of the mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD). The target range for MMAD is 1.0 to 3.0 microns and GSD is 1.5 to 3.5. The mean aerosol particle size exposure system 2, 3 and 4 were 2.1, 1.9 and 1.3 microns, respectively. Mean GSD for exposure system 2, 3 and 4 were 2.35, 2.47 and 2.15, respectively.
- Treatment of exhaust air: exhaust passed through a Solberg canister filter prior to entering the facility exhaust system, which consists of redundant exhaust blowers preceded by activated charcoal- and HEPA-filtration units.

TEST ATMOSPHERE
- Brief description of analytical method used: gravimetric
- Samples taken from breathing zone: yes

VEHICLE (if applicable)
- Justification for use and choice of vehicle: filtered air
- Composition of vehicle: not applicable
- Type and concentration of dispersant aid (if powder): not applicable
- Concentration of test material in vehicle: not applicable
- Lot/batch no. of vehicle (if required): not applicable
- Purity of vehicle: not applicable

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples were collected using pre-weighed filters held in a filter holder and placed in the animal breathing zone of an exposure system. A volume of the test atmosphere is pulled through the filter. Following sample collection, the sample is reweighed. The exposure concentration (mg/m3) were calculated by dividing the gravimetrically determined mass of test substance aerosol by the sample volume. The mass was determined by subtracting the initial filter weight from the final weight of the post-sample filter. Sample volume was calculated by multiplying the sample flow rate by the length of the sampling period. Concentrations were not be adjusted for test substance purity. For the test substance exposure systems, samples were collected daily at appropriate intervals based on the target exposure concentrations. For the control group (Group 1), gravimetric samples were collected at least twice weekly during the 13-week exposure period.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

5 days a week for 6 hours a day

Dose / conc.:

5 mg/m³ air (nominal)

Dose / conc.:

15 mg/m³ air (nominal)

Dose / conc.:

45 mg/m³ air (nominal)

Dose / conc.:

5.1 mg/m³ air (analytical)

Dose / conc.:

14.9 mg/m³ air (analytical)

Dose / conc.:

44 mg/m³ air (analytical)

No. of animals per sex per dose:

20

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Exposure concentrations were selected to cover a range extending from an anticipated no observed-adverse-effect-concentration (NOAEC) to a concentration in which toxic effects are seen in the highest exposure concentration. Specific concentrations were based on the results of a 28-day range-finding study in which microscopic findings were noted in the lungs, trachea larynx and nasal levels at a concentration of 100 mg/m3.
- Rationale for animal assignment (if not random): random
- Post-exposure recovery period in satellite groups: The animals scheduled for the recovery evaluations will be subjected to necropsy following a 4-week non-exposure period (minimum of 27 days).

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed twice daily, once in the morning and once in the afternoon. Moribund animals were euthanized and necropsied as soon as possible. Animals found dead were necropsied as soon as possible to ensure that tissues will not be lost due to autolysis.
- Cage side observations checked in table were included: mortality, abnormalities, and signs of pain and distress

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Prior to treatment, weekly during the study period and on the day of necropsy

BODY WEIGHT: Yes
- Time schedule: Prior to treatment, twice weekly during the study period and on the day of necropsy

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 / No / Not specified

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule: Once during acclimation prior to randomization and all surviving animals near the end of the exposure period or near the end of the recovery period.
- Dose groups that were examined: all animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to necropsy
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to necropsy
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table [No.1] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: on the day of necropsy
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table [No.1] were examined.

NEUROBEHAVIOURAL EXAMINATION: No
- Time schedule for examinations: not conducted, as not triggered by clinical observations

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table No 2)
HISTOPATHOLOGY: Yes (see table No 2)

Statistics:

All analyses were two-tailed for significance levels of 5% and 1%. All means were presented with standard deviations. All statistical tests were performed by a computer with appropriate programming as referenced below.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were no clinical observations attributable to treatment in the low and mid-dose groups, or in the recovery groups. There were no clinical signs noted 0-1 hour following exposure in any group.

The only group with clinical signs of toxicity due to treatment was the high dose (45 mg/m3) group during the treatment period. Males and females of this group had multiple instances of red, brown, and/or yellow material on various body surfaces and clear discharge around the eyes. The staining was judged by the study author to be indicative of test substance-related stress or general ill health. This finding was observed prior to dosing and on non-exposure days during the treatment period.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One male in the 15 mg/m3 group was euthanized in extremis on Study Day 61 due to a clinical observation of red penile discharge. At necropsy, this male was noted with gross observations of dilated pelvis, distended bladder, distended ureters, and red fluid in the bladder and ureters. This death was not considered to be test substance-related. In addition, a single male in the control group was found dead on Study Day 65; there were no remarkable clinical observations or gross observations noted for this animal. All other animals survived to the scheduled necropsies.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There were no treatment-related effects on body weight or body weight gain in the low and mid-dose groups.

Test substance-related effects on body weights were noted in the 45 mg/m3 group males and females. Lower mean body weights and cumulative body weight gains were noted beginning as early as Study Week 1 in males and females, which continued until the primary necropsy (Study Week 13); the differences were generally significant compared to the control group. On Study Week 13 (the day of the primary necropsy), mean body weights in the 45 mg/m3 males and females were 9.7% and 6.4% lower, respectively, than the mean control group body weights. In addition, body weight gains in the 45 mg/m3 group males and females were comparable or higher than the control group values during the recovery period; while mean body weights in the males remained lower (not statistically significant) than the control group. Therefore, the animals recovered once exposure stopped, but males were slightly slower to recover than females.

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:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The only treatment-related effect on organ weights occurred in the high dose group and was limited to the lungs. There were no adverse findings in the low and mid-dose groups.

At the end of the treatment period: Lung weights (absolute and relative to body and brain weights) tended to be higher with exposure concentration across all groups with statistical significance achieved only at 45 mg/m3. For the males, all group mean and individual animal absolute lung weights, as examples, were within historical control reference ranges. In contrast for the 45 mg/m3 group females, mean absolute and both mean relative weights exceeded their historical control reference ranges and, as an example, 6 individual animal absolute lung weights also exceeded the historical control reference range. The most likely microscopic correlations for the higher lung weights were the findings affecting the bronchioles (epithelial hyperplasia, mixed cell infiltrates and luminal debris). Since none of these findings were seen in either sex at exposure concentrations < 45 mg/m3, the 8% to 10% higher lung weights seen in the 15 mg/m3 group males (and lesser magnitudes of change seen at lower exposure concentrations in both sexes), which were reversible, were considered incidental and unrelated to treatment.

At the end of the recovery period: treatment-related and statistically significant higher lung weights (absolute and/or relative to body and brain weights) persisted in both sexes at exposure concentrations of 45 mg/m3. Although mean absolute lung weight was not statistically significant in the 45 mg/m3 group males, the approximately 10% increase was considered sufficient to be considered an effect of test substance exposure. Peribronchiolar chronic inflammation was considered the microscopic correlate for the persistent high lung weights in this group.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Macroscopic findings were observed in the high dose group only at the end of the treatment period and were limited to the lungs. There were no adverse findings in any of the groups at the end of the recovery period.

Treatment-related findings of not fully collapsed (3/10 males and 8/10 females) and pale lungs (1/10 males and 2/10 females) were observed exclusively in the 45 mg/m3 group at the primary necropsy; luminal debris in the bronchioles and peribronchiolar inflammatory cell infiltrates appeared to be the microscopic correlates for lungs not fully collapsed while a correlation was not observed for pale lungs. At the Study Week 17 recovery necropsy, not fully collapsed lungs
was seen in 2 males and 1 female from the 45 mg/m3 group but also 1 male each from the control and 5 mg/m3 groups. Therefore at the recovery necropsy, lungs not fully collapsed was not conclusively associated with exposure to the test substance aerosol.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

See Tables 3 to 6.
Treatment-related microscopic findings were limited to the respiratory tract. These findings were observed in all groups at the end of the treatment period, but they were only observed in the high dose group at the end of the recovery period.

At the end of the treatment period: In the nasal cavity treatment-related epithelial (transitional, respiratory, or olfactory) degeneration/regeneration was characterized by loss of normal tissue architecture, most commonly by cell loss and replacement by a single layer of attenuated epithelium; these changes were typically bilateral. Specific to the respiratory epithelium was loss of cilia and reduction/loss of goblet cells. The sites of respiratory epithelial degeneration/regeneration were most commonly the dorsal aspect of the nasal septum and lining the dorsal meatus, including nasal turbinate(s). Olfactory epithelial degeneration/regeneration was limited to 1 female in the 45 mg/m3 group and was diffuse within the dorsal meatus of nasal level III while focal and unilateral in the posterior nasal levels. Transitional epithelial degeneration/regeneration was most commonly limited to the tissues lining the lateral meatus (nasal turbinates and lateral wall). Squamous cell metaplasia and mixed cell inflammation were usually associated with areas of epithelial degeneration/regeneration. Squamous cell metaplasia represented a change from the normal epithelium lining a region to an attenuated stratified and keratinized squamous epithelium. Mixed cell inflammation was characterized by a mononuclear cell (lymphocytes, plasma cells and macrophages) population admixed with variable numbers of neutrophils. In contrast to epithelial degeneration/regeneration seen most commonly in the females, transitional and respiratory epithelial hyperplasia was the most common epithelial change seen in the males exposed to test substance aerosol; affected regions were comparable to those seen with degeneration/regeneration in the females. Several of these findings (transitional epithelial hyperplasia, mixed cell inflammation, and squamous cell metaplasia) at nasal level II were seen in one or both sexes at exposure concentrations of 5, 15, and 45 mg/m3.

In the larynx, trachea, and bronchi (recorded under lungs) treatment-related respiratory epithelial degeneration/regeneration and mixed cell inflammation were comparable to that seen in the nasal cavity. It was not uncommon in control group animals to see respiratory epithelial loss and/or attenuation at these sites. Loss was clearly an artifact of tissue collection/processing while the cause of the thinned epithelium was less certain. However, respiratory epithelial degeneration/regeneration was diagnosed only when there was single cell necrosis, mitotic figure(s) and/or rare neutrophils (sub- or intra-epithelial) associated with the attenuated epithelium. Disruption of the normal epithelial architecture with loss of cilia was also considered degeneration/regeneration. In the larynx, squamous metaplasia was similar to that
seen in the nasal cavity except that it was more commonly seen as a change from the normal non-keratinized stratified epithelium to an attenuated and keratinized stratified squamous epithelium. In addition to mixed cell inflammation in the larynx, chronic active inflammation was also an effect of exposure to the test substance, particularly in the 45 mg/m3 group females, and the epiglottis was a common site of this inflammation. Chronic active inflammation was similar to mixed cell inflammation except in the former, fibroblasts/fibroplasia were present generally indicating inflammation of longer duration. Several of these findings (respiratory epithelial
degeneration of bronchi and mixed cell inflammation of the larynx and bronchi) were seen in one or both sexes at exposure concentrations of 5, 15, and 45 mg/m3.

At the end of the recovery period: In the nasal cavity (levels II and III) most previous effects of exposure persisted in one or both sexes from the 45 mg/m3 group with
incidences and severities that were generally lower than those seen at the primary necropsy. A new effect, and evidence of chronic irritation, was mucous cell hyperplasia of the nasal septum at nasal level II in the males. Due to the low incidence and minimal to mild severity and resolution during the 4-week recovery period, findings in the nasal cavity were considered to be nonadverse at 5 and 15 mg/m3.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Details on results:

In the lungs at the primary necropsy, treatment-related effects were centered on the bronchi and/or bronchioles. Changes in the bronchi were comparable to and considered an extension of the changes seen in the trachea. The most prominent change was noted at the periphery of the lungs at the level of the terminal bronchioles. In control group animals, terminal bronchioles rarely had any associated inflammatory or mononuclear cell infiltrates and the epithelial lining
was an admixture of ciliated respiratory epithelial cells and cells with distinct apical blebbing (club/Clara cells). Exposure-related effects included variably dense perivascular and/or peribronchiolar mixed cell infiltrates characterized by mononuclear cells with generally fewer eosinophils and/or neutrophils and in some cases the suggestion of increased fibrosis/fibroplasia (Masson’s stain suggested this was true in a 45 mg/m3 group female evaluated). There was loss of the normal epithelium lining the terminal bronchioles with replacement by a prominent respiratory epithelium with a robustly ciliated epithelium; club cells were generally absent (mild respiratory epithelial hyperplasia). With minimal respiratory epithelial hyperplasia, club cells were occasionally present and/or there was epithelial alteration comparable to the respiratory epithelial degeneration/regeneration noted in the bronchi. Some animals had bronchiolar luminal debris (exudate) consisting of a mucinous material admixed with foamy alveolar macrophages and less commonly neutrophils. It was this exudate in combination with the peribronchiolar cellular infiltrates and respiratory epithelial hyperplasia that were the most likely microscopic correlations for the lungs noted at necropsy as not fully collapsed. Other than the changes affecting the bronchi, discussed above, all treatment-related effects to the lower airways were limited to the 45 mg/m3 group.

In the lungs at the recovery necropsy, some of the previous effects of exposure persisted in one or both sexes from the 45 mg/m3 group with incidences and severities that were generally lower than those seen at the primary necropsy. A new effect, and evidence of chronic irritation, was the shift from mixed cell peribronchiolar infiltrates to chronic inflammation of the bronchioles. Chronic inflammation was characterized by peribronchiolar fibrosis admixed with mononuclear cell infiltrates. Although the absence of club cells was a component of respiratory epithelial hyperplasia of the bronchioles at Study Week 13, they remained reduced in number to absent at the recovery necropsy. Aggregates of pigmented alveolar macrophages, most likely containing hemosiderin, was also a new apparent effect of test substance administration in the 45 mg/m3 group males. Two males from the 45 mg/m3 group had proliferative changes characterized as ‘atypical’ including bronchiolo-alveolar hyperplasia and bronchial mucous cell metaplasia.

Key result

Dose descriptor:

NOAEC

Effect level:

ca. 15 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

45 mg/m³ air (nominal)

System:

other: whole respiratory tract

Organ:

larynx

lungs

nasal cavity

trachea

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Table 3 Incidence of Selected Histopathologic Findings, Study Week 13 Primary Necropsy - males

	Males
Exposure Concentration (mg/m^3)	0	5	15	45
Nasal level II^a	11^b	10	11^b	10
Degeneration/regeneration, respiratory epithelium	0	0	0	2
Minimal	-	-	-	2
Mild	-	-	-	0
Moderate	-	-	-	0
Degeneration/regeneration, transitional epithelium	0	0	0	0
Minimal	-	-	-	-
Mild	-	-	-	-
Moderate	-	-	-	-
Marked	-	-	-	-
Hyperplasia, respiratory epithelium	0	0	0	6
Minimal	-	-	-	4
Mild	-	-	-	2
Hyperplasia, transitional epithelium	1	4	6^b	3
Minimal	1	4	6^b	2
Mild	0	0	0	1
Inflammation, mixed cell	0	2	5	10
Minimal	-	2	4	10
Mild	-	0	1	0
Metaplasia, squamous cell	0	1	4	6
Minimal	-	1	4	4
Mild	-	0	0	2
Nasal level III^a	11^b	NA	NA	10
Degeneration/regeneration, olfactory epithelium (marked)	0	-	-	0
Degeneration/regeneration, respiratory epithelium	0	-	-	1
Minimal	-	-	-	1
Mild	-	-	-	0
Larynx^a	11^b	10	10^b	10
Degeneration/regeneration, respiratory epithelium(minimal)	0	0	1	4
Inflammation, chronic active	0	0	2	1
Minimal	-	-	0	0
Mild	-	-	2	1
Inflammation, mixed cell	2	2	5^b	6
Minimal	2	2	3^b	4
Mild	0	0	2	2
Metaplasia, squamous cell	0	0	0	2
Minimal	-	-	-	1
Mild	-	-	-	1
Trachea^a	11^b	9	11^b	10
Degeneration/regeneration, respiratory epithelium	0	0	3^b	7
Minimal	-	-	2^b	5
Mild	-	-	1	2
Moderate	-	-	0	0
Inflammation, mixed cell (minimal)	1^b	0	1	3
Lungs^a	11^b	10	11^b	10
Degeneration/regeneration, respiratory epithelium, bronchus	0	4	9^b	7
Minimal	-	4	9^b	5
Mild	-	0	0	2
Hyperplasia, respiratory epithelium, bronchioles	0	0	0	7
Minimal	-	-	-	6
Mild	-	-	-	1
Infiltrate, mixed cell, peribronchiolar	0	0	0	7
Minimal	-	-	-	3
Mild	-	-	-	4
Inflammation, mixed cell, bronchus	0	3	3	4
Minimal	-	3	3	3
Mild	-	0	0	1
Luminal debris, bronchioles	0	0	0	2
Minimal	-	-	-	0
Mild	-	-	-	2

^a Number of tissues examined from each group.

^b Includes 1 FD/EE rat.

BOLDED incidences represent effects of exposure to N-[3-(trimethoxysilyl)propyl]ethylenediamine, CAS 1760-24-3

Table 4 Incidence of Selected Histopathologic Findings, Study Week 13 Primary Necropsy - females

	Females
Exposure Concentration (mg/m^3)	0	5	15	45
Nasal level II^a	10	10	10	10
Degeneration/regeneration, respiratory epithelium	0	0	0	8
Minimal	-	-	-	2
Mild	-	-	-	2
Moderate	-	-	-	4
Degeneration/regeneration, transitional epithelium	0	0	1	8
Minimal	-	-	1	1
Mild	-	-	0	1
Moderate	-	-	0	2
Marked	-	-	0	4
Hyperplasia, respiratory epithelium	0	0	0	0
Minimal	-	-	-	-
Mild	-	-	-	-
Hyperplasia, transitional epithelium	0	0	0	0
Minimal	-	-	-	-
Mild	-	-	-	-
Inflammation, mixed cell	2	5	4	8
Minimal	2	3	4	3
Mild	0	2	0	5
Metaplasia, squamous cell	0	0	1	4
Minimal	-	-	0	4
Mild	-	-	1	0
Nasal level III^a	10	10	10	10
Degeneration/regeneration, olfactory epithelium (marked)	0	0	0	1
Degeneration/regeneration, respiratory epithelium	0	0	0	3
Minimal	-	-	-	1
Mild	-	-	-	2
Larynx^a	10	10	10	10
Degeneration/regeneration, respiratory epithelium(minimal)	0	0	1	1
Inflammation, chronic active	0	0	0	5
Minimal	-	-	-	2
Mild	-	-	-	3
Inflammation, mixed cell	1	4	7	7
Minimal	1	4	5	6
Mild	0	0	2	1
Metaplasia, squamous cell	0	0	0	5
Minimal	-	-	-	5
Mild	-	-	-	0
Trachea^a	10	10	10	10
Degeneration/regeneration, respiratory epithelium	0	0	2	8
Minimal	-	-	1	3
Mild	-	-	1	4
Moderate	-	-	0	1
Inflammation, mixed cell (minimal)	0	0	1	5
Lungs^a	10	10	10	10
Degeneration/regeneration, respiratory epithelium, bronchus	0	1	5	9
Minimal	-	1	5	7
Mild	-	0	0	2
Hyperplasia, respiratory epithelium, bronchioles	0	0	0	10
Minimal	-	-	-	10
Mild	-	-	-	0
Infiltrate, mixed cell, peribronchiolar	0	0	0	10
Minimal	-	-	-	5
Mild	-	-	-	5
Inflammation, mixed cell, bronchus	0	1	0	6
Minimal	-	1	-	6
Mild	-	0	-	0
Luminal debris, bronchioles	0	0	0	7
Minimal	-	-	-	3
Mild	-	-	-	4

^a Number of tissues examined from each group.

^b Includes 1 FD/EE rat.

BOLDEDincidences represent effects of exposure to N-[3-(trimethoxysilyl)propyl]ethylenediamine, CAS 1760-24-3

Table 5 Incidence of Selected Histopathologic Findings, Study Week 17 Recovery Necropsy - males

	Males
Exposure Concentration (mg/m^3)	0	5	15	45
Nasal level II^a	9	10	9	10
Degeneration/regeneration, respiratory epithelium(minimal)	0	0	0	1
Degeneration/regeneration, transitional epithelium	0	0	0	1
Minimal	-	-	-	1
Mild	-	-	-	0
Hyperplasia, mucous cell (minimal)	0	1	1	5
Hyperplasia, respiratory epithelium (mild)	0	0	0	2
Inflammation, mixed cell	2	1	1	3
Minimal	2	1	1	1
Mild	0	0	0	2
Nasal level III^a	NA	NA	NA	NA
Degeneration/regeneration, olfactory epithelium (moderate)	-	-	-	-
Inflammation, mixed cell (mild)	-	-	-	-
Larynx^a	9	10	9	10
Degeneration/regeneration, respiratory epithelium(minimal)	0	0	0	0
Inflammation, chronic	0	0	0	4
Minimal	-	-	-	1
Mild	-	-	-	3
Trachea^a	9	10	9	10
Degeneration/regeneration, respiratory epithelium (Minimal)	0	0	0	0
Inflammation, mixed cell (minimal)	0	0	0	0
Lungs^a	9	10	9	10
Decreased club cells, terminal bronchioles (Present)	0	0	0	8
Degeneration/regeneration, respiratory epithelium, bronchus (Minimal)	0	0	0	0
Inflammation, chronic, bronchioles (Minimal)	0	0	0	7
Luminal debris, bronchioles (Minimal)	0	0	0	2
Macrophages, pigmented (Minimal)	0	1	0	3

^a Number of tissues examined from each group.

BOLDEDincidences represent effects of exposure to N-[3-(trimethoxysilyl)propyl]ethylenediamine,

CAS 1760-24-3

There were no other test substance-related histologic changes. Remaining histologic changes were considered to be incidental findings or related to some aspect of experimental manipulation other than exposure to N-[3-(trimethoxysilyl)propyl]ethylenediamine. There was no test substance-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Table 6 Incidence of Selected Histopathologic Findings, Study Week 17 Recovery Necropsy - females

	Females
Exposure Concentration (mg/m^3)	0	5	15	45
Nasal level II^a	10	10	10	10
Degeneration/regeneration, respiratory epithelium(minimal)	0	0	0	1
Degeneration/regeneration, transitional epithelium	0	0	0	2
Minimal	-	-	-	0
Mild	-	-	-	2
Hyperplasia, mucous cell (minimal)	0	0	0	0
Hyperplasia, respiratory epithelium (mild)	0	0	0	0
Inflammation, mixed cell	2	0	1	5
Minimal	2	-	1	4
Mild	0	-	0	1
Nasal level III^a	10	10	10	10
Degeneration/regeneration, olfactory epithelium (moderate)	0	0	0	1
Inflammation, mixed cell (mild)	0	0	0	1
Larynx^a	10	10	10	10
Degeneration/regeneration, respiratory epithelium(minimal)	0	0	0	3
Inflammation, chronic	0	0	0	6
Minimal	-	-	-	3
Mild	-	-	-	3
Trachea^a	10	10	10	10
Degeneration/regeneration, respiratory epithelium (Minimal)	0	0	0	1
Inflammation, mixed cell (minimal)	0	0	0	1
Lungs^a	10	10	10	10
Decreased club cells, terminal bronchioles (Present)	0	0	0	10
Degeneration/regeneration, respiratory epithelium, bronchus (Minimal)	0	0	0	1
Inflammation, chronic, bronchioles (Minimal)	0	0	0	9
Luminal debris, bronchioles (Minimal)	0	0	0	5
Macrophages, pigmented (Minimal)	0	0	0	0

^a Number of tissues examined from each group.

BOLDEDincidences represent effects of exposure to N-[3-(trimethoxysilyl)propyl]ethylenediamine,

CAS 1760-24-3

There were no other test substance-related histologic changes. Remaining histologic changes were considered to be incidental findings or related to some aspect of experimental manipulation other than exposure to N-[3-(trimethoxysilyl)propyl]ethylenediamine. There was no test substance-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Table 7 Correlation of Selected Observations

 

Necropsy	Organ Weight	Clinical Pathology	Histopathology
Lung- not fully collapsed (PN; 45 mg/m^3)	¿ lung weight	-	Lung - Luminal debris, bronchioles; respiratory epithelial hyperplasia of the bronchioles and mixed cell infiltrate, peribronchiolar
-	¿ lung weight (RN;  45 mg/m^3)	-	Lung- Luminal debris, bronchioles and chronic inflammation, bronchioles

 

 Relationships were suspected between gross necropsy, organ weight, clinical pathology, and histopathology observations, as presented in Table 7. These proposed relationships were based on subjective interpretation rather than a statistical analysis of correlation.

Conclusions:

In a 90-day repeated dose toxicity study conducted according to OECD Test Guideline 413 and in compliance with GLP, exposure of Sprague-Dawley rats to N-[3-(trimethoxysilyl)propyl] ethylenediamine via nose-only inhalation for 6 hours per day on a 5-day basis for 13 weeks at target exposure concentrations of 5, 15, and 45 mg/m3 resulted in non-adverse test substance-related effects at 5 and 15 mg/m3. Adverse test substance-related clinical observations, lower body weights and body weight gains, macroscopic findings in the lungs, increased lung weights, and microscopic findings in the nasal cavity, larynx, trachea, and lungs were noted in the 45 mg/m3 group males and females at the end of the treatment period. At the recovery necropsy, many of the effects of test substance exposure, including higher lung weights, persisted in the nasal cavity, larynx, trachea, and lungs in the 45 mg/m3 group and were noted at generally a lower incidence and severity than that seen at the primary necropsy. Therefore, the NOAEC was concluded to be 15 mg/m3.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

15 mg/m³

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

This study was designed to determine the effects in rats resulting from repeated percutaneous exposure to A-1120 for 9 doses over an 11 day period.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Type of coverage:

occlusive

Vehicle:

unchanged (no vehicle)

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

11 days

Frequency of treatment:

9 applications

Dose / conc.:

257.5 mg/kg bw/day (nominal)

Dose / conc.:

772.5 mg/kg bw/day (nominal)

Dose / conc.:

1 545 mg/kg bw/day (nominal)

No. of animals per sex per dose:

20/sex/control and high dose
10/sex/ mid and low dose

Control animals:

other: filtered water

Dose descriptor:

NOAEL

Effect level:

>= 1 545 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No signs of systemic toxicity.

Critical effects observed:

no

No clinical signs of toxicity except local effects on skin. Barely perceptible erythema was observed occasionally in males of the 772.5 and 1545 mg/kg bw/day groups and in females of the 1545 mg/kg bw/day group during the first week of treatment. Exfoliation and/or excoriation, were observed during the treatment period in males and females of the 772.5 and 1545 mg/kg bw/day groups. One female of the 257.5 mg/kg bw/day group also showed excoriation during the treatment period. During the 19 -day recovery period, exfoliation and/or excoriation were observed in males and females of the 1545 mg/kg bw/day group. No skin reactions were observed after day 17.

Decreases in food consumption were observed in males of the 772.5 and 1545 mg/kg bw/day during the treatment period. Some body weight decreases were observed but there was no clear dose-response. Slight increases in water consumption were observed in females of the 772.5 and 1545 mg/kg bw/day groups during the study. However, the increases were not considered to be toxicologically significant due to the lack of any associated changes in clinical pathology parameters and the small magnitude of the changes. There were no treatment-related effects on haematology, clinical chemistry, isoenzymes or urinalysis at the end of the treatment period. There was no effect on haematological parameters at the end of the recovery period.

There were slight increases in adrenal weights in the mid and high dose groups, but in light of the lack of pathological findings these were not considered to be toxicologically significant.

Various signs of skin irritation were observed. Exfoliation and excoriation were noted at the end of the treatment period of males in the mid and high dose groups, and females of all groups. Microscopic findings in these groups were hyperkeratosis, acanthosis, epidermitis and dermatitis. Ulceration and dermal fibrosis were observed occasionally in the same treated groups. Residual effects, as indicated by minimal hyperkeratosis and dermatitis were observed in males and females of the high dose group at the end of the 19 -day recovery period.

Conclusions:

Treatment of rats to N-(3-(trimethoxysilyl)propyl)ethylenediamine for nine cutaneous applications during an 11-day period produced transient clinical, necropsy and microscopic observations indicative of mild to moderate skin irritation in males treated with 772.5 and 1545 mg/kg bw/day and females treated with 257.5 mg/kg bw/day and above. There was no clear indication of systemic toxicity. Therefore the NOAEL for systemic effects is at least 1545 mg/kg bw/day in rats.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 545 mg/kg bw/day

Study duration:

subacute

Species:

rat

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

This study was designed to determine the effects in rats resulting from repeated percutaneous exposure to A-1120 for 9 doses over an 11 day period.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Type of coverage:

occlusive

Vehicle:

unchanged (no vehicle)

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

11 days

Frequency of treatment:

9 applications

Dose / conc.:

257.5 mg/kg bw/day (nominal)

Dose / conc.:

772.5 mg/kg bw/day (nominal)

Dose / conc.:

1 545 mg/kg bw/day (nominal)

No. of animals per sex per dose:

20/sex/control and high dose
10/sex/ mid and low dose

Control animals:

other: filtered water

Dose descriptor:

NOAEL

Effect level:

>= 1 545 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No signs of systemic toxicity.

Critical effects observed:

no

No clinical signs of toxicity except local effects on skin. Barely perceptible erythema was observed occasionally in males of the 772.5 and 1545 mg/kg bw/day groups and in females of the 1545 mg/kg bw/day group during the first week of treatment. Exfoliation and/or excoriation, were observed during the treatment period in males and females of the 772.5 and 1545 mg/kg bw/day groups. One female of the 257.5 mg/kg bw/day group also showed excoriation during the treatment period. During the 19 -day recovery period, exfoliation and/or excoriation were observed in males and females of the 1545 mg/kg bw/day group. No skin reactions were observed after day 17.

Decreases in food consumption were observed in males of the 772.5 and 1545 mg/kg bw/day during the treatment period. Some body weight decreases were observed but there was no clear dose-response. Slight increases in water consumption were observed in females of the 772.5 and 1545 mg/kg bw/day groups during the study. However, the increases were not considered to be toxicologically significant due to the lack of any associated changes in clinical pathology parameters and the small magnitude of the changes. There were no treatment-related effects on haematology, clinical chemistry, isoenzymes or urinalysis at the end of the treatment period. There was no effect on haematological parameters at the end of the recovery period.

There were slight increases in adrenal weights in the mid and high dose groups, but in light of the lack of pathological findings these were not considered to be toxicologically significant.

Various signs of skin irritation were observed. Exfoliation and excoriation were noted at the end of the treatment period of males in the mid and high dose groups, and females of all groups. Microscopic findings in these groups were hyperkeratosis, acanthosis, epidermitis and dermatitis. Ulceration and dermal fibrosis were observed occasionally in the same treated groups. Residual effects, as indicated by minimal hyperkeratosis and dermatitis were observed in males and females of the high dose group at the end of the 19 -day recovery period.

Conclusions:

Treatment of rats to N-(3-(trimethoxysilyl)propyl)ethylenediamine for nine cutaneous applications during an 11-day period produced transient clinical, necropsy and microscopic observations indicative of mild to moderate skin irritation in males treated with 772.5 and 1545 mg/kg bw/day and females treated with 257.5 mg/kg bw/day and above. There was no clear indication of systemic toxicity. Therefore the NOAEL for systemic effects is at least 1545 mg/kg bw/day in rats.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

257.5 mg/cm²

Study duration:

subacute

Species:

rat

Additional information

In a 28-day dose range-finding study, which was not conducted according to an OECD guideline or in compliance with GLP, 3 male and 3 female rats were administered daily oral (gavage) doses of 0, 50, 100, 250, 500 and 750 mg/kg bw/day N-[3 -(dimethoxymethylsilyl)propyl]ethylenediamine (CAS No. 3069-29-2, EC No. 221-336-6) in corn oil over a period of 28 days. Treatment with the test item resulted in moderately increased mean ALAT (alamine aminotransferase) and severely increased mean ASAT (aspartate aminotransferase) levels for test item-treated males at 750 mg/kg bw/day when compared to the control group. No clinical signs or mortality could clearly be related to treatment with the test item up to 750 mg/kg bw/day. No haematological parameters, no gross macroscopic lesions or toxicologically relevant differences in organ weights were observed. Based on the data generated from this dose range finding study, dose levels of 100, 300, and 1000 mg/kg bw/day are selected for the subsequent main study with N-[3 -(dimethoxymethylsilyl)propyl]ethylenediamine (BSL Bioservice, 2020).

In the key 90 -day oral repeated dose toxicity conducted according to OECD Test Guideline 408 and in compliance with GLP, 10 males and 10 females rats per group were administered daily oral (gavage) doses of 0, 100, 300 and 1000 mg/kg bw/day N-[3-(dimethoxymethylsilyl)propyl]ethylenediamine (CAS 3069-29-2; EC 221-336-6) in corn oil on 7 days per week. Due to the irritating nature of the test substance formulation and mortality in the High Dose (HD) group, the dose level of the high dose group was reduced twice in the course of the treatment period, first reduced to 750 mg/kg bw/day and finally reduced to 500 mg/kg bw/day.

There were 5 additional animals added to the control and high dose group which served as a recovery group and were observed for an additional 4-week treatment-free period. During the study, there were 21 unscheduled deaths, which included one female of Low Dose (LD) group, one male and two females of Middle Dose (MD), and 9 males and 8 females of the HD group.

In decedents of the LD and HD groups, necrotic and/or inflammatory lesions were observed in the respiratory tract, as well as in the pleural region and/or esophagus. These lesions were considered to be due to incidental influx or gavage error during administration of the test item formulation with its irritating nature. There were also findings suggesting gavage error of the test item formulation in the MD decedents, however, autolysis/postmortem changes had been advanced in these animals. Necrotic and inflammatory lesions in the organs and tissues mentioned above were most probably involved in the animals’ death and morbidity, but any findings were deemed not to be due to a systemic effect of the test item.

In decedents of the HD group, necrotic and/or inflammatory lesions were also present in the stomach. These were considered to be a local tissue injury associated with the irritating nature of the test item and were likely involved in the animals’ death and morbidity. In survivors, there was no irritative effect leading to lethal tissue injury in the stomach and respiratory tract.

In this study, microscopic changes that could be attributed to a systemic effect of the test item were observed in the kidney of both decedents and survivors. These included proximal tubular cell vacuolation, proximal tubular cell necrosis, and increased incidence and/or severity of tubular basophilia and tubular dilatation. These renal lesions were considered to correlate with a significant increase or increased tendency in kidney weights of the HD group. In animals necropsied at the end of the recovery period, proximal tubular vacuolation and necrosis were not found in any HD animals, and there were no qualitative differences in the other renal findings between the control and HD groups. There were also no differences in kidney weights in both sexes. Thus, the histopathological examination revealed that the treatment-related renal changes completely subsided during the recovery phase of this study. In the liver, centrilobular hepatocellular hypertrophy was observed in decedents of the HD group. There were no further indicators of tissue and cell injury, and therefore, it was deemed to be of an adaptive nature. Hepatocellular hypertrophy was not found in any HD animals that survived until the end of the treatment period after the dose level had been reduced twice during the course of the treatment period.

In conclusion, based on the absence of the treatment-related renal lesions in the survivors of the MD group under the condition of this study, the no-observed-adverse-effect-level (NOAEL) may be established at 300 mg/kg bw/day for both sexes. Further, the histopathological examination suggests that a dose level less than 500 mg/kg bw/day of this test item formulation would not produce irritative tissue injury if it was competently administered by gavage without error into the stomach (BSL Bioservice, 2021).

Repeated dose toxicity supporting information for the analogue substance N-(3-(trimethoxysilyl)propyl)ethylenediamine (CAS No. 1760-24-3, EC No. 217-164-6) are included only to support the read-across justification for the genetic toxicity endpoint currently using this analogue substance.

A supporting oral repeated dose toxicity study was conducted according to OECD Test Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test) with the analogue substance N-(3-(trimethoxysilyl)propyl)ethylenediamine.

There were three unscheduled deaths during the conduct of the study. None of the unscheduled deaths were attributed to the test substance.

Clinical findings attributed to test substance included clear perioral soiling in several high dose animals and either increased nasal sounds, laboured respiration, or soft vocalizations in approximately half of the high dose females and one high dose male. These signs were not seen in the control animals and infrequently seen in either of the two lower dose groups. Observations recorded at dosing indicated a dose-related resistance to dosing. Evaluating all 30 animals/dose (males, female toxicity and female reproductive groups) over the entire dosing period, the incidence of resistance was 3, 5, 27 and 62 for the controls, 25, 125 and 500 mg/kg bw/day dose groups, respectively. Similar incidence patterns were noted for salivation just prior to dosing, wetness around the mouth at dosing, and wetness around the mouth 5-30 minutes following dosing.

There were no test substance-related effects on body weight or food consumption for any of the dose groups. No test substance-related changes on FOB and Motor activity parameters were observed in the male and female toxicity animals evaluated. There were no test substance-related changes in haematology and serum chemistry parameters for these animals. No test substance-related effects were observed at the macroscopic examinations for any of the animals (males, females toxicity, female reproductive or the pups). There were no effects on mean organ weights or organ to body weight ratios attributable to the test substance for the selected organs evaluated (adrenal glands, brain, heart, kidneys, liver, spleen, thymus, uterus, ovaries, testes, epididymides, prostate, and seminal vesicles) in the male and toxicity female animals.

A thorough histopathological examination performed on all gross lesions, selected tissues and organs for control and high dose toxicity group animals demonstrated no microscopic findings directly attributable to test substance treatment. No test substance-related effects were observed in any of the reproductive parameters evaluated. Two high dose (500 mg/kg bw/day) and one low dose (25 mg/kg bw/day) reproductive group females that did not produce litters had positive evidence of copulation. Six of the eight surviving high dose reproductive group females produced litters that were similar in all respects to control litters.

In this study, the systemic NOAEL for the analogue substance N-(3-(trimethoxysilyl)propyl)ethylenediamine was concluded to be greater than or equal to 500 mg/kg bw/day.

In the 90-day repeated dose inhalation toxicity study conducted according to OECD Test Guideline 413 and in compliance with GLP, exposure of Sprague-Dawley rats to an aerosol of the analogue substance N-[3-(trimethoxysilyl)propyl] ethylenediamine via nose-only inhalation for 6 hours per day on a 5-day basis for 13 weeks at target exposure concentrations of 5, 15, and 45 mg/m³ resulted in non-adverse test substance-related effects at 5 and 15 mg/m³ (Charles River, 2017a). Adverse test substance-related clinical observations, lower body weights and body weight gains, macroscopic findings in the lungs, increased lung weights, and microscopic findings in the nasal cavity, larynx, trachea, and lungs were noted in the 45 mg/m³ group males and females at the end of the treatment period. At the recovery necropsy, many of the effects of test substance exposure, including higher lung weights, persisted in the nasal cavity, larynx, trachea, and lungs in the 45 mg/m³ group and were noted at generally a lower incidence and severity than that seen at the primary necropsy. Therefore, the NOAEC for the local effects on the lungs and respiratory tract was 15 mg/m³. There were no adverse systemic effects observed at any concentration.

In a dose-range-finding study, rats were exposed to respirable liquid aerosol of the analogue substance (N-[3-(trimethoxysilyl)propyl]ethylenediamine) at target exposure concentrations of 10, 50,100, and 500 mg/m³ via 6-hour nose-only inhalation exposures 5 days per week for 4 weeks. The NOAEC from this study was 10 mg/m³ based on local respiratory effects (Charles River, 2017b).

Treatment of rats with the analogue substance N-(3-(trimethoxysilyl)propyl)ethylenediamine for nine cutaneous applications during an 11-day period produced transient clinical, necropsy and microscopic observations indicative of mild to moderate skin irritation in males treated with 772.5 and 1545 mg/kg bw/day, and females treated with 257.5 mg/kg bw/day and above. There was no clear indication of systemic toxicity. Therefore the NOAEL for systemic effects is concluded to be greater than or equal to 1545 mg/kg bw/day in rats. The LOAEL for dermal local effects was established at 257.5 mg/kg bw/day (Bushy Run Research Centre, 1993).

Justification for classification or non-classification

Based on the available information, N-(3-(dimethoxymethylsilyl)propyl)ethylenediamine is not classified for target organ toxicity effects following repeated exposures according to Regulation (EC) No. 1272/2008.